Production of chemicals from renewable sources

ABSTRACT

Among other things, the present disclosure provides biosynthesis polypeptides, methods, and non-naturally occurring microbial organisms for preparing various compounds such as 1,5-pentanediol, adipic acid, 1,6-hexanediol, 6-hydroxy hexanoic acid, and 2-keto carboxylic acids.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a National Stage Entry of PCT/US2020/029981, filed Apr. 25, 2020, which claims priority to United States Provisional Application Nos. 62/838,793, filed Apr. 25, 2019, and 62/868,824, filed Jun. 28, 2019, the entirety of each of which is incorporated herein by reference.

SEQUENCE LISTING

The present application contains a Sequence Listing, which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jun. 17, 2022, is named SequenceListing.txt and is 320,465 bytes in size.

TECHNICAL FIELD

This disclosure relates generally to compositions and methods of preparation of industrially useful chemicals.

BACKGROUND

Adipic acid (AA) is a widely used chemical with an estimated 2.3 million metric tons demand in 2012 (IHS Chemical, Process Economics Program Report: Bio-Based Adipic Acid (December 2012)). Along with hexamethylenediamine (HMDA), it is used in the production of nylon6,6, polyester resins, plasticizers, foods, and other materials. Thus, methods of preparing adipic acid in high yield using renewable sources are highly desirable.

1,5-Pentanediol is a major component of polyurethanes and polyesters (PDL). 1,6-Hexanediol (HDO), is a linear diol with terminal hydroxyl groups. It is used in polyesters for industrial coating applications, two-component polyurethane coatings for automotive applications. It is also used for production of macrodiols for example adipate esters and polycarbonate diols used in elastomers and polyurethane dispersions for parquet flooring and leather coatings.

6-Hydroxy hexanoic acid (6HH) can be cyclized to make ε-caprolactone which can then be aminated to make ε-caprolactam. ε-Caprolactam is used for the production of Nylon6, a widely used polymer in many different industries. ε-Caprolactone is polymerized to make polycaprolactone (PCL) a biodegradable polyester with applications for the production of specialty polyurethanes.

2-Keto carboxylic acids are useful intermediates for the preparation of a number of industrially relevant chemicals and pharmaceutical drugs. They are precursors for production of amino acids, as well as industrially useful α-hydroxy carboxylic acids.

SUMMARY

Among other things, the present disclosure encompasses the recognition that certain biosynthesis peptides, e.g., various enzymes, can be utilized to efficiently prepare various compounds, in many embodiments, from substrates that are structurally different from their natural and/or characterized substrates. In some embodiments, the present disclosure provides technologies (e.g., enzymes, nucleic acids, organisms, cultures, etc.) for preparing various compounds utilizing one or more such enzymes.

For example, in some embodiments, the present disclosure provides that aldol-dehydration product biosynthesis polypeptides, such as various hydratase-aldolases, can be effectively utilized to prepare a number of compounds from aliphatic aldehydes other than their typical aromatic aldehyde substrates. In some embodiments, the present disclosure provides a method comprising:

contacting pyruvate and an aliphatic aldehyde with an aldol-dehydration product biosynthesis polypeptide so that an aldol-dehydration product is produced, wherein:

the carbonyl group of the aliphatic aldehyde is not conjugated to a alkenyl, alkynyl, or aromatic group; and

the aldol-dehydration product is a compound comprising an aldehyde or ketone group and a double bond conjugated with the aldehyde or ketone group.

In some embodiments, an aldehyde, e.g., an aliphatic aldehyde has the structure of formula A-1:

R^(a)-L²-L¹-C(O)H,   A-1

or a salt thereof, wherein:

R^(a) is R″ or —OR″,

each of L¹ and L² is independently a covalent bond, or a bivalent, optionally substituted, linear or branched C₁₋₂₀ aliphatic or C₁₋₂₀ heteroaliphatic, wherein one or more methylene units are optionally and independently replaced by —C≡C—, —C(R″)₂—, -Cy-, —O—, —S—, —S—S—, —N(R″)—, —C(O)—, —C(S)—, —C(NR″)—, —C(O)N(R″)—, —N(R″)C(O)N(R″)—, —N(R″)C(O)O—, —S(O)—, —S(O)₂—, —S(O)₂N(R″)—, —C(O)S—, or —C(O)O—;

-Cy- is a bivalent, optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring, wherein each monocyclic ring is independently an optionally substituted, saturated, partially saturated or aromatic 3-20 membered ring having 0-5 heteroatoms;

each R″ is independently —R′, —C(O)R′, —CO₂R′, or —SO₂R′;

R′ is hydrogen, or an optionally substituted group selected from C₁₋₁₀ aliphatic, C₁₋₁₀ heteroaliphatic having 1-5 heteroatoms, a 6-10 membered aryl ring, a 5-10 membered heteroaryl ring having 1-5 heteroatoms, and a 3-10 membered heterocyclic ring having 1-5 heteroatoms, or:

two or more R′ groups are taken together with their intervening atoms to form an optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring having, in addition to the intervening atoms, 0-5 heteroatoms, wherein each monocyclic ring is independently an optionally substituted, saturated, partially saturated or aromatic 3-20 membered ring having 0-5 heteroatoms.

In some embodiments, L¹ is optionally substituted —CH₂—. In some embodiments, L¹ is optionally monosubstituted —CH₂—. In some embodiments, L¹ is —CH₂—.

In some embodiments, an aldol-dehydration product has the structure of formula P-2:

R^(a)-L²-L¹-CH═CH—C(O)—C(O)OH,   P-2

or a salt thereof, wherein each variable is independently as described herein.

As described herein, an aldol-dehydration product, e.g., a compound of formula P-2 or a salt thereof, can be further processed, in some embodiments, through one or more biosynthetic processes to provide various products, such as 1,5-pentanediol, HDO, 6HH, adipic acid, etc. (e.g., see FIGS. 2-5) and various products made therefrom, including various polymeric products made therefrom.

In some embodiments, as shown herein, an aldol-dehydration product, e.g., a compound of formula P-2 or a salt thereof may also be prepared from an aldol product, e.g., a compound of formula P-1:

R^(a)-L²-L¹-CH(OH)—CH₂—C(O)—C(O)OH,   P-1

or a salt thereof, wherein each variable is independently as described herein.

In some embodiments, an aldol-dehydration product is manufactured by contacting an aldol product with a dehydration product biosynthesis polypeptide.

In some embodiments, an aldol product is manufactured by contacting suitable substrates with an aldol product biosynthesis polypeptide.

In some embodiments, the present disclosure demonstrates that various alkene reduction product biosynthesis polypeptides can be utilized to manufacture various compounds from their natural or non-natural substrates. In some embodiments, the present disclosure provides a method comprising:

contacting an alkene with an alkene reduction product biosynthesis polypeptide so that an alkene reduction product is produced, wherein:

the alkene comprises a double bond conjugated to a carbonyl group; and

a double bond conjugated to a carbonyl group in the alkene is reduced to a single bond to provide an alkene reduction product.

In some embodiments, an alkene is an aldol-dehydration product, e.g. one of formula P-2 or a salt thereof. In some embodiments, an alkene reduction product has the structure of formula P-3:

R^(a)-L²-L¹-CH₂—CH₂—C(O)—C(O)OH,   P-3

or a salt thereof, wherein each variable is independently as described herein.

Among other things, disclosed herein are enzymes, methods, and recombinant microorganisms for preparing 2-keto carboxylic acids, 1,5-pentanediol, adipic acid, 1,6-hexanediol, and 6-hydroxy hexanoic acid using renewable sources.

In one aspect, provided herein is a method for producing a 2-keto carboxylic acid of formula:

wherein R is H, CH₃, or CH₂OH; the method comprising or consisting essentially of contacting pyruvate and

with a hydratase-aldolase and a quinone oxidoreductase in a culture or organisms comprising one or more non-naturally occurring microorganisms to produce the 2-keto carboxylic acid; wherein the hydratase-aldolase and the quinone oxidoreductase are expressed by the one or more non-naturally occurring microorganisms.

In another aspect, provided herein is a method for producing a 2-keto carboxylic acid of formula:

wherein R is H, CH₃, or CH₂OH; the method comprising or consisting essentially of contacting pyruvate and

with a hydratase-aldolase and a quinone oxidoreductase in a culture or organisms comprising two or more non-naturally occurring microorganisms to produce the 2-keto carboxylic acid; wherein the hydratase-aldolase and the quinone oxidoreductase are expressed by the two or more non-naturally occurring microorganisms.

In another aspect, provided herein is a method for producing 1,5-pentanediol, the method comprising or consisting essentially of,

-   contacting pyruvate and 3-hydroxy-propanal with a hydratase-aldolase     and a quinone oxidoreductase to produce a 2-keto carboxylic acid of     formula:

wherein R is CH₂OH;

-   contacting the 2-keto carboxylic acid with a     2-keto-acid-decarboxylase to produce a 5-hydroxy-pentanal; and -   contacting the 5-hydroxy-pentanal with a primary alcohol     dehydrogenase to produce the 1,5-pentanediol, wherein the method is     performed in a culture comprising one or more non-naturally     occurringmicrobial organisms.

In another aspect, provided herein is a method for producing 1,5-pentanediol, the method comprising or consisting essentially of,

-   contacting pyruvate and 3-hydroxy-propanal with a hydratase-aldolase     and a quinone oxidoreductase to produce a 2-keto carboxylic acid of     formula:

wherein R is CH₂OH;

-   contacting the 2-keto carboxylic acid with a     2-keto-acid-decarboxylase to produce a 5-hydroxy-pentanal; and -   contacting the 5-hydroxy-pentanal with a primary alcohol     dehydrogenase to produce the 1,5-pentanediol, wherein the method is     performed in a culture comprising two or more non-naturally     occurringmicrobial organisms.

In another aspect, provided herein is a method for producing 1,6-hexanediol, the method comprising

-   contacting pyruvate and 3-hydroxy-propanal with a hydratase-aldolase     and a quinone oxidoreductase to produce a 2-keto carboxylic acid of     formula:

wherein R is CH₂OH;

-   contacting the 2-keto carboxylic acid with a     6-hydroxy-2-oxohexanoate-2-reductase to produce     2,6-dihydroxy-hexanoate; -   contacting the 2,6-dihydroxy-hexanoate with a     2,6-dihydroxy-hexanoate CoA-transferase to produce     2,6-dihydroxy-hexanoyl-CoA; -   contacting the 2,6-dihydroxy-hexanoyl-CoA with a the     2,6-dihydroxy-hexanoyl-CoA 2-dehydratase to produce     6-hydroxy-2,3-dehydro-hexanoyl-CoA; -   contacting the 6-hydroxy-2,3-dehydro-hexanoyl-CoA with a     2,3-dehydro-hexanoyl-CoA 2,3-reductase to produce     6-hydroxy-hexanoyl-CoA; -   contacting the 6-hydroxy-hexanoyl-CoA with a 6-hydroxyhexanoyl-CoA     transferase to produce 6-hydroxy-hexanoate; -   contacting the 6-hydroxy-hexanoate with a 6-hydroxyhexanoate     1-reductase to produce 6-hydroxy-hexanal; and -   contacting the 6-hydroxy-hexanal with a 6-hydroxyhexanal 1-reductase     to produce the 1,6-hexanediol, wherein the method is performed in a     culture comprising one or more non-naturally occurring microbial     organisms.

In another aspect, provided herein is a method for producing 1,6-hexanediol, the method comprising

-   contacting pyruvate and 3-hydroxy-propanal with a hydratase-aldolase     and a quinone oxidoreductase to produce a 2-keto carboxylic acid of     formula:

wherein R is CH₂OH;

-   contacting the 2-keto carboxylic acid with a     6-hydroxy-2-oxohexanoate-2-reductase to produce     2,6-dihydroxy-hexanoate; -   contacting the 2,6-dihydroxy-hexanoate with a     2,6-dihydroxy-hexanoate CoA-transferase to produce     2,6-dihydroxy-hexanoyl-CoA; -   contacting the 2,6-dihydroxy-hexanoyl-CoA with a the     2,6-dihydroxy-hexanoyl-CoA 2-dehydratase to produce     6-hydroxy-2,3-dehydro-hexanoyl-CoA; -   contacting the 6-hydroxy-2,3-dehydro-hexanoyl-CoA with a     2,3-dehydro-hexanoyl-CoA 2,3-reductase to produce     6-hydroxy-hexanoyl-CoA; -   contacting the 6-hydroxy-hexanoyl-CoA with a 6-hydroxyhexanoyl-CoA     transferase to produce 6-hydroxy-hexanoate; -   contacting the 6-hydroxy-hexanoate with a 6-hydroxyhexanoate     1-reductase to produce 6-hydroxy-hexanal; and -   contacting the 6-hydroxy-hexanal with a 6-hydroxyhexanal 1-reductase     to produce the 1,6-hexanediol, wherein the method is performed in a     culture comprising two or more non-naturally occurring microbial     organisms.

In another aspect, provided herein is a method for producing 6-hydroxy-hexanoate, the method comprising

-   contacting pyruvate and 3-hydroxy-propanal with a hydratase-aldolase     and a quinone oxidoreductase to produce a 2-keto carboxylic acid of     formula:

wherein R is CH₂OH;

-   contacting the 2-keto carboxylic acid with a     6-hydroxy-2-oxohexanoate-2-reductase to produce     2,6-dihydroxy-hexanoate; -   contacting the 2,6-dihydroxy-hexanoate with a     2,6-dihydroxy-hexanoate CoA-transferase to produce     2,6-dihydroxy-hexanoyl-CoA; -   contacting the 2,6-dihydroxy-hexanoyl-CoA with a the     2,6-dihydroxy-hexanoyl-CoA 2-dehydratase to produce     6-hydroxy-2,3-dehydro-hexanoyl-CoA; -   contacting the 6-hydroxy-2,3-dehydro-hexanoyl-CoA with a     2,3-dehydro-hexanoyl-CoA 2,3-reductase to produce     6-hydroxy-hexanoyl-CoA; and -   contacting the 6-hydroxy-hexanoyl-CoA with a 6-hydroxyhexanoyl-CoA     transferase to produce the 6-hydroxy-hexanoate;     wherein the method is performed in a culture comprising one or more     non-naturally occurring microbial organisms.

In another aspect, provided herein is a method for producing 6-hydroxy-hexanoate, the method comprising

-   contacting pyruvate and 3-hydroxy-propanal with a hydratase-aldolase     and a quinone oxidoreductase to produce a 2-keto carboxylic acid of     formula:

wherein R is CH₂OH;

-   contacting the 2-keto carboxylic acid with a     6-hydroxy-2-oxohexanoate-2-reductase to produce     2,6-dihydroxy-hexanoate; -   contacting the 2,6-dihydroxy-hexanoate with a     2,6-dihydroxy-hexanoate CoA-transferase to produce     2,6-dihydroxy-hexanoyl-CoA; -   contacting the 2,6-dihydroxy-hexanoyl-CoA with a the     2,6-dihydroxy-hexanoyl-CoA 2-dehydratase to produce     6-hydroxy-2,3-dehydro-hexanoyl-CoA; -   contacting the 6-hydroxy-2,3-dehydro-hexanoyl-CoA with a     2,3-dehydro-hexanoyl-CoA 2,3-reductase to produce     6-hydroxy-hexanoyl-CoA; and -   contacting the 6-hydroxy-hexanoyl-CoA with a 6-hydroxyhexanoyl-CoA     transferase to produce the 6-hydroxy-hexanoate;     wherein the method is performed in a culture comprising two or more     non-naturally occurring microbial organisms.

In another aspect, provided herein is a method for producing adipic acid, the method comprising

-   contacting pyruvate and 3-hydroxy-propanal with a hydratase-aldolase     and a quinone oxidoreductase to produce a 2-keto carboxylic acid of     formula:

wherein R is CH₂OH;

-   contacting the 2-keto carboxylic acid with a     6-hydroxy-2-oxohexanoate-2-reductase to produce     2,6-dihydroxy-hexanoate; -   contacting the 2,6-dihydroxy-hexanoate with a     2,6-dihydroxy-hexanoate CoA-transferase to produce     2,6-dihydroxy-hexanoyl-CoA; -   contacting the 2,6-dihydroxy-hexanoyl-CoA with a the     2,6-dihydroxy-hexanoyl-CoA 2-dehydratase to produce     6-hydroxy-2,3-dehydro-hexanoyl-CoA; -   contacting the 6-hydroxy-2,3-dehydro-hexanoyl-CoA with a     2,3-dehydro-hexanoyl-CoA 2,3-reductase to produce     6-hydroxy-hexanoyl-CoA; -   contacting the 6-hydroxy-hexanoyl-CoA with a 6-hydroxyhexanoyl-CoA     transferase to produce 6-hydroxy-hexanoate; -   contacting the 6-hydroxy-hexanoate with a 6-hydroxyhexanoate     dehydrogenase to produce 6-oxo-hexanoate; and -   contacting the 6-oxo-hexanoate with a 6-oxo-hexanoate oxidase to     produce the adipic acid,     wherein the method is performed in a culture comprising one or more     non-naturally occurring microbial organisms.

In another aspect, provided herein is a method for producing adipic acid, the method comprising

-   contacting pyruvate and 3-hydroxy-propanal with a hydratase-aldolase     and a quinone oxidoreductase to produce a 2-keto carboxylic acid of     formula:

wherein R is CH₂OH;

-   contacting the 2-keto carboxylic acid with a     6-hydroxy-2-oxohexanoate-2-reductase to produce     2,6-dihydroxy-hexanoate; -   contacting the 2,6-dihydroxy-hexanoate with a     2,6-dihydroxy-hexanoate CoA-transferase to produce     2,6-dihydroxy-hexanoyl-CoA; -   contacting the 2,6-dihydroxy-hexanoyl-CoA with a the     2,6-dihydroxy-hexanoyl-CoA 2-dehydratase to produce     6-hydroxy-2,3-dehydro-hexanoyl-CoA; -   contacting the 6-hydroxy-2,3-dehydro-hexanoyl-CoA with a     2,3-dehydro-hexanoyl-CoA 2,3-reductase to produce     6-hydroxy-hexanoyl-CoA; -   contacting the 6-hydroxy-hexanoyl-CoA with a 6-hydroxyhexanoyl-CoA     transferase to produce 6-hydroxy-hexanoate; -   contacting the 6-hydroxy-hexanoate with a 6-hydroxyhexanoate     dehydrogenase to produce 6-oxo-hexanoate; and -   contacting the 6-oxo-hexanoate with a 6-oxo-hexanoate oxidase to     produce the adipic acid,     wherein the method is performed in a culture comprising two or more     non-naturally occurring microbial organisms.

In some embodiments, the hydratase-aldolase is an enzyme having an EC number 4.1.2.45, EC number 4.1.2.34 or EC number 4.1.1.4. In some embodiments, the hydratase-aldolase is an enzyme selected from the group of enzymes identified under GenBank, RefSeq, or Uniprot ID Nos. D7C0E5, P0A144, Q79EM8, A0A0N0AHI8, A0A0N1FRY3, M3DYR1, W7SU48, A0A286PH18, Q9X9Q6, Q9WXH7, A4XDS1, F2J6N9, A0A063BFL5, Q9ZHH6, A0A0C1K853, WP_034398482, PYK12191, WP_115478033, WP_028222253, WP_013654807, WP_059403060, WP_092508530, WP_116642627, WP_009770659, WP_107818191, WP_003292061, PYN48855, WP_122212965, WP_028217297, WP_034507049, KMK64081.1, WP_070028041.1, or KZL92449.1.

In some embodiments, the hydratase-aldolase is an enzyme having an EC number 4.1.2.45, EC number 4.1.2.34 or EC number 4.1.1.4. In some embodiments, the hydratase-aldolase is an enzyme selected from the group of enzymes identified under GenBank, RefSeq, or Uniprot ID Nos. D7C0E5, P0A144, Q79EM8, A0A0N0AHI8, A0A0N1FRY3, M3DYR1, W7SU48, A0A286PH18, Q9X9Q6, Q9WXH7, A4XDS1, F2J6N9, A0A063BFL5, Q9ZHH6, A0A0C1K853, WP_034398482, PYK12191, A0A370X7D8, WP_028222253, F2J6L6, A0A0N0L9F6, A0A1G9YWG7, A0A2U1BT09, A0A244DHE8, WP_107818191, A0A023WZF9, PYN48855, A0A421PAQ6, WP_028217297, WP_034507049, KMK64081.1, WP_070028041.1, or KZL92449.1. In some embodiments, the hydratase-aldolase is an enzyme comprising a sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:84, SEQ ID NO:85, or SEQ ID NO:86.

In some embodiments, the hydratase-aldolase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme selected from the group of enzymes identified under GenBank, RefSeq, or Uniprot ID Nos. D7C0E5, P0A144, Q79EM8, A0A0N0AHI8, A0A0N1FRY3, M3DYR1, W7SU48, A0A286PH18, Q9X9Q6, Q9WXH7, A4XDS1, F2J6N9, A0A063BFL5, Q9ZHH6, A0A0C1K853, WP_034398482, PYK12191, A0A370X7D8, WP_028222253, F2J6L6, A0A0N0L9F6, A0A1G9YWG7, A0A2U1BT09, A0A244DHE8, WP_107818191, A0A023WZF9, PYN48855, A0A421PAQ6, WP_028217297, WP_034507049, KMK64081.1, WP_070028041.1, or KZL92449.1, or a portion (e.g., a domain, a set of amino acid residues (can be continuous or separated), etc.) thereof that promotes the formation of a aldol-dehydration product. In some embodiments, the hydratase-aldolase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme comprising a sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:84, SEQ ID NO:85, or SEQ ID NO:86.

In some embodiments, the hydratase-aldolase is an enzyme selected from Tables 1 and 5-8. In some embodiments, the hydratase-aldolase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme selected from Tables 1 and 5-8.

In some embodiments, the quinone oxidoreductase is an enzyme having an EC number 1.6.5. In some embodiments, the quinone oxidoreductase is an enzyme having an EC number 1.6.5.5. In some embodiments, the quinone oxidoreductase is an enzyme selected from the group of enzymes identified under GenBank, RefSeq, or Uniprot ID Nos. P28304, P40783, Q0K2I0, A0A1Z1SRY9, P43903, 17G8G0, or Q142L2, ALK19324.1, A0A1G9R408, G4Q8R5, ANA98723.1, K0EUQ3, A0A061CRS8, Q9A212, A0A1I6RWW2, WP_026197277.1, Q5NKZ3, WP_012333034.1, or WP_136898000.1. In some embodiments, the quinone oxidoreductase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme selected from the group of enzymes identified under GenBank, RefSeq, or Uniprot ID Nos. P28304, P40783, Q0K2I0, A0A1Z1SRY9, P43903, I7G8G0, or Q142L2, ALK19324.1, A0A1G9R408, G4Q8R5, ANA98723.1, K0EUQ3, A0A061CRS8, Q9A212, A0A1I6RWW2, WP_026197277.1, Q5NKZ3, WP_012333034.1, or WP_136898000.1. In some embodiments, the quinone oxidoreductase is an enzyme comprising a sequence of SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, or SEQ ID NO:97. In some embodiments, the quinone oxidoreductase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme comprising a sequence of SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, or SEQ ID NO:97.

In some embodiments, the hydratase-aldolase and the quinone oxidoreductase are expressed by the one or more non-naturally occurring microbial organisms. In some embodiments, at least one of the hydratase-aldolase and the quinone oxidoreductase enzymes are expressed by one or more exogenous genes expressed by the one or more non-naturally occurring microorganisms. In some embodiments, the hydratase-aldolase is exogenously expressed by the one or more non-naturally occurring microorganisms. In some embodiments, the quinone oxidoreductase is exogenously expressed by the one or more non-naturally occurring microbial organisms. In some embodiments, the quinone oxidoreductase is overexpressed by the one or more non-naturally occurring microbial organisms. In some embodiments, the hydratase-aldolase is exogenously expressed by the one or more non-naturally occurring microbial organisms and the quinone oxidoreductase is overexpressed by the one or more non-naturally occurring microbial organisms.

In some embodiments, the hydratase-aldolase and the quinone oxidoreductase are expressed by the two or more non-naturally occurring microbial organisms. In some embodiments, at least one of the hydratase-aldolase and the quinone oxidoreductase enzymes are expressed by one or more exogenous genes expressed by the two or more non-naturally occurring microorganisms. In some embodiments, the hydratase-aldolase is exogenously expressed by the two or more non-naturally occurring microorganisms. In some embodiments, the quinone oxidoreductase is exogenously expressed by the two or more non-naturally occurring microbial organisms. In some embodiments, the quinone oxidoreductase is overexpressed by the one or more non-naturally occurring microbial organisms. In some embodiments, the hydratase-aldolase is exogenously expressed by the two or more non-naturally occurring microbial organisms and the quinone oxidoreductase is overexpressed by the two or more non-naturally occurring microbial organisms.

In some embodiments, one or more of the hydratase-aldolase and quinone oxidoreductase further comprise one or more protein tags. In some embodiments, the protein tags are selected from polyhistidine tag, a GST tag (glutathione-S-transferase tag), a HA tag (hemagglutinin tag), a FLAG tag, a Myc tag, a maltose binding protein tag, a chitin binding protein tag, and a fluorescent tag.

In some embodiments, the method for producing a 2-keto carboxylic acid further comprises or consists essentially of separating the 2-keto carboxylic acid from the one or more non-naturally occurring microbial organisms or a culture comprising the one or more non-naturally occurring microbial organisms. In some embodiments, the method further comprises or consists essentially of separating the 2-keto carboxylic acid from the two or more non-naturally occurring microbial organisms or a culture comprising the two or more non-naturally occurring microbial organisms.

In some embodiments, the 2-keto-acid-decarboxylase is an enzyme selected from the group of enzymes identified under an EC number 4.1.1.1; EC number 4.1.1.2; EC number 4.1.1.3; EC number 4.1.1.4; EC number 4.1.1.5; EC number 4.1.1.6; EC number 4.1.1.7; EC number 4.1.1.11; EC number 4.1.1.12; EC number 4.1.1.15; EC number 4.1.1.16; EC number 4.1.1.17; EC number 4.1.1.18; EC number 4.1.1.19; EC number 4.1.1.20; EC number 4.1.1.34; EC number 4.1.1.35; EC number 4.1.1.40; EC number 4.1.1.54; EC number 4.1.1.56; EC number 4.1.1.71; EC number 4.1.1.72; EC number 4.1.1.73; EC number 4.1.1.74; EC number 4.1.1.75; or EC number 4.1.1.77. In some embodiments, the 2-keto-acid-decarboxylase is an enzyme selected from the group of enzymes identified under Uniprot ID No. Q6QBS4, A7M7D6, or P20906. In some embodiments, the 2-keto-acid-decarboxylase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme selected from the group of enzymes identified under Uniprot ID No. Q6QBS4, A7M7D6, or P20906.

In some embodiments, the primary alcohol dehydrogenase is an enzyme having an EC number 1.1.1.61. In some embodiments, the primary alcohol dehydrogenase is an enzyme selected from the group of enzymes identified under Uniprot or GenBank ID Nos. NP_417279.1, NP_349892.1, NP 349891.1, BAB12273.1, L21902.1, Q94B07, AAB03015.1, NP_014032.1, NP_013892.1, NP_015019.1, NP_010996.2, ABX39192.1, XP_001210625.1, ABO67118, ABO68223, BAE77068.1, or CAA47743.1. In some embodiments, the primary alcohol dehydrogenase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme selected from the group of enzymes identified under Uniprot or GenBank ID Nos. NP_417279.1, NP_349892.1, NP_349891.1, BAB12273.1, L21902.1, Q94B07, AAB03015.1, NP_014032.1, NP_013892.1, NP_015019.1, NP_010996.2, ABX39192.1, XP_001210625.1, ABO67118, ABO68223, BAE77068.1, or CAA47743.1. In some embodiments, the primary alcohol dehydrogenase is an enzyme comprising a sequence of SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, or SEQ ID NO:74. In some embodiments, the primary alcohol dehydrogenase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme comprising a sequence of SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, or SEQ ID NO:74.

In some embodiments, the hydratase-aldolase is an enzyme identified under Uniprot ID No. A0A286PH18; the quinone oxidoreductase is an enzyme identified under Uniprot ID No. P28304; the 2-keto-acid-decarboxylase is an enzyme identified under Uniprot ID No. Q6QBS4; and the primary alcohol dehydrogenase is an enzyme identified under Uniprot or GenBank ID Nos. D6Z860, YP_001705436.1, ANO06407.1, AAR91681.1, AHH98121.1, ANB00612.1, ANO04655.1, A0R484, AFP42026.1, GAJ86510.1, YP_001704097.1, ANA99315.1, GAJ83027.1, ANA98925.1, ANA98924.1, ANO04656.1, YP_001703694. In some embodiments, the hydratase-aldolase is an enzyme comprising a sequence of SEQ ID NO:8; the quinone oxidoreductase is an enzyme comprising a sequence of SEQ ID NO:45; the 2-keto-acid-decarboxylase is an enzyme comprising a sequence of SEQ ID NO:83; and the primary alcohol dehydrogenase is an enzyme comprising a sequence of SEQ ID NO:70.

In some embodiments, the 2-keto-acid-decarboxylase and the primary alcohol dehydrogenase are expressed by the one or more non-naturally occurring microbial organisms. In some embodiments, the 2-keto-acid-decarboxylase and the primary alcohol dehydrogenase are exogenously expressed by the one or more non-naturally occurring microbial organisms.

In some embodiments, the 2-keto-acid-decarboxylase and the primary alcohol dehydrogenase are expressed by the two or more non-naturally occurring microbial organisms. In some embodiments, the 2-keto-acid-decarboxylase and the primary alcohol dehydrogenase are exogenously expressed by the two or more non-naturally occurring microbial organisms.

In some embodiments, one or more of the hydratase-aldolase, quinone oxidoreductase, 2-keto-acid-decarboxylase, and primary alcohol dehydrogenase further comprise one or more protein tags. In some embodiments, the protein tags are selected from polyhistidine tag, a GST tag (glutathione-S-transferase tag), a HA tag (hemagglutinin tag), a FLAG tag, a Myc tag, a maltose binding protein tag, a chitin binding protein tag, and a fluorescent tag.

In some embodiments, the method for producing a 1,5-pentanediol further comprises or consists essentially of separating the 1,5-pentanediol from the one or more non-naturally occurring microbial organisms or a culture comprising the one or more non-naturally occurring microbial organisms. In some embodiments, the method further comprises or consists essentially of separating the 1,5-pentanediol from the two or more non-naturally occurring microbial organisms or a culture comprising the two or more non-naturally occurring microbial organisms.

In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase, the 2,6-dihydroxy-hexanoate CoA-transferase, the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase, the 2,3-dehydro-hexanoyl-CoA 2,3-reductase, the 6-hydroxyhexanoyl-CoA transferase, the 6-hydroxyhexanoate 1-reductase, and the 6-hydroxyhexanal 1-reductase are expressed by the one or more non-naturally occurring microbial organisms. In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase, the 2,6-dihydroxy-hexanoate CoA-transferase, the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase, the 2,3-dehydro-hexanoyl-CoA 2,3-reductase, the 6-hydroxyhexanoyl-CoA transferase, the 6-hydroxyhexanoate 1-reductase, and the 6-hydroxyhexanal 1-reductase are exogenously expressed by the one or more non-naturally occurring microbial organisms.

In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase, the 2,6-dihydroxy-hexanoate CoA-transferase, the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase, the 2,3-dehydro-hexanoyl-CoA 2,3-reductase, the 6-hydroxyhexanoyl-CoA transferase, the 6-hydroxyhexanoate 1-reductase, and the 6-hydroxyhexanal 1-reductase are expressed by the two or more non-naturally occurring microbial organisms. In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase, the 2,6-dihydroxy-hexanoate CoA-transferase, the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase, the 2,3-dehydro-hexanoyl-CoA 2,3-reductase, the 6-hydroxyhexanoyl-CoA transferase, the 6-hydroxyhexanoate 1-reductase, and the 6-hydroxyhexanal 1-reductase are exogenously expressed by the two or more non-naturally occurring microbial organisms.

In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase is an enzyme selected from the group of enzymes identified under an EC number 1.1.99.6, EC number 1.1.1.169, EC number 1.1.1.215, EC number 1.1.1.28, or EC number 1.1.1.110; the 2,6-dihydroxy-hexanoate CoA-transferase is an enzyme selected from the group of enzymes identified under an EC number 2.8.3, EC number 2.8.3.1, or EC number 2.8.3.12; the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase is an enzyme having an EC number 4.2.1.167; the 2,3-dehydro-hexanoyl-CoA 2,3-reductase is an enzyme having an EC number 1.3.1.44; the 6-hydroxyhexanoyl-CoA transferase is an enzyme having an EC number 2.8.3, EC number 2.8.3.1, or EC number 2.8.3.12; the 6-hydroxyhexanoate 1-reductase is an enzyme having an EC number 1.2.99.6; and the 6-hydroxyhexanal 1-reductase is an enzyme having an EC number 1.1.1.

In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase is an enzyme selected from the group of enzymes identified under Uniprot or GenBank ID Nos. WP_003431407.1, BAL51292.1, Q5FTU6, AKC64094.1, WP_002876862.1, AGP69017.1, WP_003640741.1, AKC64095.1, and AKC64094.1; the 2,6-dihydroxy-hexanoate CoA-transferase is an enzyme selected from the group of enzymes identified under Uniprot ID No. T4VW93; the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase is an enzyme selected from the group of enzymes identified under Uniprot ID Nos. Q5U924, Q5U925, and Q5U923; the 2,3-dehydro-hexanoyl-CoA 2,3-reductase is an enzyme identified under Uniprot ID No. Q73Q47; the 6-hydroxyhexanoyl-CoA transferase is an enzyme identified under Uniprot ID No. T4VW93; the 6-hydroxyhexanoate 1-reductase is an enzyme identified under Uniprot or GenBank ID Nos. D6Z860, YP_001705436.1, ANO06407.1, AAR91681.1, AHH98121.1, ANB00612.1, ANO04655.1, A0R484, AFP42026.1, GAJ86510.1, YP_001704097.1, ANA99315.1, GAJ83027.1, ANA98925.1, ANA98924.1, ANO04656.1, YP_001703694.1, WP_036338301.1, WP_007472106.1, or A0QWI7; and the 6-hydroxyhexanal 1-reductase is an enzyme identified under Uniprot or GenBank ID Nos. D6Z860, YP_001705436.1, ANO06407.1, AAR91681.1, AHH-98121.1, ANB00612.1, ANO04655.1, A0R484, AFP42026.1, GAJ86510.1, YP_001704097.1, ANA99315.1, GAJ83027.1, ANA98925.1, ANA98924.1, ANO04656.1, YP_001703694.

In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase is an enzyme comprising a sequence of SEQ ID NO:53, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, or SEQ ID NO:105; the 2,6-dihydroxy-hexanoate CoA-transferase is an enzyme comprising a sequence of SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, or SEQ ID NO:58; the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase is an enzyme comprising a sequence of SEQ ID NO:59, SEQ ID NO:61, and SEQ ID NO:63; or SEQ ID NO:60, SEQ ID NO:62, and SEQ ID NO:64; the 2,3-dehydro-hexanoyl-CoA 2,3-reductase is an enzyme comprising a sequence of SEQ ID NO:65; the 6-hydroxyhexanoyl-CoA transferase is an enzyme comprising a sequence of SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, or SEQ ID NO:58; the 6-hydroxyhexanoate 1-reductase is an enzyme comprising a sequence of SEQ ID NO:66, SEQ ID NO:67, or SEQ ID NO:68; and the 6-hydroxyhexanal 1-reductase is an enzyme comprising a sequence of SEQ ID NO:70.

In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme comprising a sequence of SEQ ID NO:53, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, or SEQ ID NO:105; the 2,6-dihydroxy-hexanoate CoA-transferase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme comprising a sequence of SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, or SEQ ID NO:58; the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme comprising a sequence of SEQ ID NO:59, SEQ ID NO:61, and SEQ ID NO:63; or SEQ ID NO:60, SEQ ID NO:62, and SEQ ID NO:64; the 2,3-dehydro-hexanoyl-CoA 2,3-reductase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme comprising a sequence of SEQ ID NO:65; the 6-hydroxyhexanoyl-CoA transferase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme comprising a sequence of SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, or SEQ ID NO:58; the 6-hydroxyhexanoate 1-reductase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme comprising a sequence of SEQ ID NO:66, SEQ ID NO:67, or SEQ ID NO:68; and the 6-hydroxyhexanal 1-reductase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme comprising a sequence of SEQ ID NO:70.

In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase is an enzyme identified under Uniprot or GenBank ID Nos. WP_003431407.1, BAL51292.1, Q5FTU6, AKC64094.1, WP_002876862.1, AGP69017.1, WP_003640741.1, AKC64095.1, and AKC64094.1; the 2,6-dihydroxy-hexanoate CoA-transferase is an enzyme identified under Uniprot ID Nos. T4VW93, A0A0C7GD16, A0A175L1W4, or A0A2X3BTQ9; the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase is an enzyme identified under Uniprot ID Nos. Q5U924, Q5U925, and Q5U923; or A0A2X3BK09, A0A2X3BU19, and A0A1V9IXA9; the 2,3-dehydro-hexanoyl-CoA 2,3-reductase is an enzyme identified under Uniprot ID No. Q73Q47; the 6-hydroxyhexanoyl-CoA transferase is an enzyme identified under Uniprot ID No. T4VW93, A0A0C7GD16, A0A175L1W4, or A0A2X3BTQ9; the 6-hydroxyhexanoate 1-reductase is an enzyme identified under Uniprot or GenBank ID Nos D6Z860, YP_001705436.1, ANO06407.1, AAR91681.1, AHH-98121.1, ANB00612.1, ANO04655.1, A0R484, AFP42026.1, GAJ86510.1, YP_001704097.1, ANA99315.1, GAJ83027.1, ANA98925.1, ANA98924.1, ANO04656.1, YP_001703694.1, WP_036338301.1, WP_007472106.1, or A0QWI7; and the 6-hydroxyhexanal 1-reductase is an enzyme identified under Uniprot or GenBank ID Nos. D6Z860, YP_001705436.1, ANO06407.1, AAR91681.1, AHH-98121.1, ANB00612.1, ANO04655.1, A0R484, AFP42026.1, GAJ86510.1, YP_001704097.1, ANA99315.1, GAJ83027.1, ANA98925.1, ANA98924.1, ANO04656.1, YP_001703694.

In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme identified under Uniprot or GenBank ID Nos. WP_003431407.1, BAL51292.1, Q5FTU6, AKC64094.1, WP_002876862.1, AGP69017.1, WP_003640741.1, AKC64095.1, and AKC64094.1; the 2,6-dihydroxy-hexanoate CoA-transferase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme identified under Uniprot ID Nos. T4VW93, A0A0C7GD16, A0A175L1W4, or A0A2X3BTQ9; the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme identified under Uniprot ID Nos. Q5U924, Q5U925, and Q5U923; or A0A2X3BK09, A0A2X3BU19, and A0A1V9IXA9; the 2,3-dehydro-hexanoyl-CoA 2,3-reductase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme identified under Uniprot ID No. Q73Q47; the 6-hydroxyhexanoyl-CoA transferase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme identified under Uniprot ID No. T4VW93, A0A0C7GD16, A0A175L1W4, or A0A2X3BTQ9; the 6-hydroxyhexanoate 1-reductase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme identified under Uniprot or GenBank ID Nos D6Z860, YP_001705436.1, ANO06407.1, AAR91681.1, AHH98121.1, ANB00612.1, ANO04655.1, A0R484, AFP42026.1, GAJ86510.1, YP_001704097.1, ANA99315.1, GAJ83027.1, ANA98925.1, ANA98924.1, ANO04656.1, YP_001703694.1, WP_036338301.1, WP_007472106.1, or A0QWI7; and the 6-hydroxyhexanal 1-reductase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme identified under Uniprot or GenBank ID Nos. D6Z860, YP_001705436.1, ANO06407.1, AAR91681.1, AHH98121.1, ANB00612.1, ANO04655.1, A0R484, AFP42026.1, GAJ86510.1, YP_001704097.1, ANA99315.1, GAJ83027.1, ANA98925.1, ANA98924.1, ANO04656.1, YP001703694.

In some embodiments, one or more of the 6-hydroxy-2-oxohexanoate-2-reductase, the 2,6-dihydroxy-hexanoate CoA-transferase, the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase, the 2,3-dehydro-hexanoyl-CoA 2,3-reductase, the 6-hydroxyhexanoyl-CoA transferase, the 6-hydroxyhexanoate 1-reductase, and the 6-hydroxyhexanal 1-reductase further comprise one or more protein tags. In some embodiments, the protein tags are selected from polyhistidine tag, a GST tag (glutathione-S-transferase tag), a HA tag (hemagglutinin tag), a FLAG tag, a Myc tag, a maltose binding protein tag, a chitin binding protein tag, and a fluorescent tag.

In some embodiments, the method for producing 1,6-hexanediol further comprises or consists essentially of separating the 1,6-hexanediol from the one or more non-naturally occurring microbial organisms or a culture comprising the one or more non-naturally occurring microbial organisms. In some embodiments, the method further comprises or consists essentially of separating the 1,6-hexanediol from the two or more non-naturally occurring microbial organisms or a culture comprising the two or more non-naturally occurring microbial organisms.

In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase, the 2,6-dihydroxy-hexanoate CoA-transferase, the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase, the 2,3-dehydro-hexanoyl-CoA 2,3-reductase, and the 6-hydroxyhexanoyl-CoA transferase are expressed by the one or more non-naturally occurring microbial organisms. In some embodiments, 6-hydroxy-2-oxohexanoate-2-reductase, the 2,6-dihydroxy-hexanoate CoA-transferase, the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase, the 2,3-dehydro-hexanoyl-CoA 2,3-reductase, and the 6-hydroxyhexanoyl-CoA transferase are exogenously expressed by the one or more non-naturally occurring microbial organisms.

In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase, the 2,6-dihydroxy-hexanoate CoA-transferase, the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase, the 2,3-dehydro-hexanoyl-CoA 2,3-reductase, and the 6-hydroxyhexanoyl-CoA transferase are expressed by the two or more non-naturally occurring microbial organisms. In some embodiments, 6-hydroxy-2-oxohexanoate-2-reductase, the 2,6-dihydroxy-hexanoate CoA-transferase, the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase, the 2,3-dehydro-hexanoyl-CoA 2,3-reductase, and the 6-hydroxyhexanoyl-CoA transferase are exogenously expressed by the two or more non-naturally occurring microbial organisms.

In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase is an selected from the group of enzymes identified under an EC number 1.1.99.6, EC number 1.1.1.169, EC number 1.1.1.215, EC number 1.1.1.28, or EC number 1.1.1.110; the 2,6-dihydroxy-hexanoate CoA-transferase is an enzyme having an EC number 2.8.3, EC number 2.8.3.1, or EC number 2.8.3.12; the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase is an enzyme having an EC number 4.2.1.167; the 2,3-dehydro-hexanoyl-CoA 2,3-reductase is an enzyme having an EC number 1.3.1.44; and the 6-hydroxyhexanoyl-CoA transferase is an enzyme having an EC number 2.8.3, EC number 2.8.3.1, or EC number 2.8.3.12.

In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase is an enzyme selected from the group of enzymes identified under Uniprot or GenBank ID Nos. WP_003431407.1, BAL51292.1, Q5FTU6, AKC64094.1, WP_002876862.1, AGP69017.1, WP_003640741.1, AKC64095.1, and AKC64094.1; the 2,6-dihydroxy-hexanoate CoA-transferase is an enzyme identified under Uniprot ID Nos. T4VW93, A0A2X3BTQ9, A0A0C7GD16, or A0A175L1W4; the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase is an enzyme identified under Uniprot ID Nos. Q5U924, Q5U925, and Q5U923; or A0A2X3BK09, A0A2X3BU19, and A0A1V9IXA9; the 2,3-dehydro-hexanoyl-CoA 2,3-reductase is an enzyme identified under Uniprot ID No. Q73Q47; and the 6-hydroxyhexanoyl-CoA transferase is an enzyme identified under Uniprot ID Nos. T4VW93, A0A2X3BTQ9, A0A0C7GD16, or A0A175L1W4.

In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme identified under Uniprot or GenBank ID Nos. WP_003431407.1, BAL51292.1, Q5FTU6, AKC64094.1, WP_002876862.1, AGP69017.1, WP_003640741.1, AKC64095.1, and AKC64094.1; the 2,6-dihydroxy-hexanoate CoA-transferase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme identified under Uniprot ID Nos. T4VW93, A0A2X3BTQ9, A0A0C7GD16, or A0A175L1W4; the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme identified under Uniprot ID Nos. Q5U924, Q5U925, and Q5U923; or A0A2X3BK09, A0A2X3BU19, and A0A1V9IXA9; the 2,3-dehydro-hexanoyl-CoA 2,3-reductase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme identified under Uniprot ID No. Q73Q47; and the 6-hydroxyhexanoyl-CoA transferase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme identified under Uniprot ID Nos. T4VW93, A0A2X3BTQ9, A0A0C7GD16, or A0A175L1W4.

In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase is an enzyme comprising a sequence of SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, or SEQ ID NO:105; the 2,6-dihydroxy-hexanoate CoA-transferase is an enzyme comprising a sequence of SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, or SEQ ID NO:58; the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase is an enzyme comprising a sequence of SEQ ID NO:59, SEQ ID NO:61, and SEQ ID NO:63; or SEQ ID NO:60, SEQ ID NO:62, and SEQ ID NO:64; the 2,3-dehydro-hexanoyl-CoA 2,3-reductase is an enzyme comprising a sequence of SEQ ID NO:65; and the 6-hydroxyhexanoyl-CoA transferase is an enzyme comprising a sequence of SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, or SEQ ID NO:58.

In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme comprising a sequence of SEQ ID NO:5, SEQ ID NO:54, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, or SEQ ID NO:105; the 2,6-dihydroxy-hexanoate CoA-transferase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme comprising a sequence of SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, or SEQ ID NO:58; the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme comprising a sequence of SEQ ID NO:59, SEQ ID NO:61, and SEQ ID NO:63; or SEQ ID NO:60, SEQ ID NO:62, and SEQ ID NO:64; the 2,3-dehydro-hexanoyl-CoA 2,3-reductase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme comprising a sequence of SEQ ID NO:65; and the 6-hydroxyhexanoyl-CoA transferase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme comprising a sequence of SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, or SEQ ID NO:58.

In some embodiments, one or more of the 6-hydroxy-2-oxohexanoate-2-reductase, the 2,6-dihydroxy-hexanoate CoA-transferase, the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase, the 2,3-dehydro-hexanoyl-CoA 2,3-reductase, and the 6-hydroxyhexanoyl-CoA transferase further comprise one or more protein tags. In some embodiments, the protein tags are selected from polyhistidine tag, a GST tag (glutathione-S-transferase tag), a HA tag (hemagglutinin tag), a FLAG tag, a Myc tag, a maltose binding protein tag, a chitin binding protein tag, and a fluorescent tag.

In some embodiments, the method for producing a 6-hydroxy-hexanoate further comprises or consists essentially of separating the 6-hydroxy-hexanoate from the one or more non-naturally occurring microbial organisms or a culture comprising the one or more non-naturally occurring microbial organisms. In some embodiments, the method further comprises or consists essentially of separating the 6-hydroxy-hexanoate from the two or more non-naturally occurring microbial organisms or a culture comprising the two or more non-naturally occurring microbial organisms.

In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase, the 2,6-dihydroxy-hexanoate CoA-transferase, the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase, the 2,3-dehydro-hexanoyl-CoA 2,3-reductase, the 6-hydroxyhexanoyl-CoA transferase, the 6-hydroxyhexanoate dehydrogenase, and the 6-oxo-hexanoate oxidase are expressed by the one or more non-naturally occurring microbial organisms. In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase, the 2,6-dihydroxy-hexanoate CoA-transferase, the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase, the 2,3-dehydro-hexanoyl-CoA 2,3-reductase, the 6-hydroxyhexanoyl-CoA transferase, the 6-hydroxyhexanoate dehydrogenase, and the 6-oxo-hexanoate oxidase are exogenously expressed by the one or more non-naturally occurring microbial organisms.

In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase, the 2,6-dihydroxy-hexanoate CoA-transferase, the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase, the 2,3-dehydro-hexanoyl-CoA 2,3-reductase, the 6-hydroxyhexanoyl-CoA transferase, the 6-hydroxyhexanoate dehydrogenase, and the 6-oxo-hexanoate oxidase are expressed by the two or more non-naturally occurring microbial organisms. In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase, the 2,6-dihydroxy-hexanoate CoA-transferase, the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase, the 2,3-dehydro-hexanoyl-CoA 2,3-reductase, the 6-hydroxyhexanoyl-CoA transferase, the 6-hydroxyhexanoate dehydrogenase, and the 6-oxo-hexanoate oxidase are exogenously expressed by the two or more non-naturally occurring microbial organisms.

In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase is an selected from the group of enzymes identified under an EC number 1.1.99.6, EC number 1.1.1.169, EC number 1.1.1.215, EC number 1.1.1.28, or EC number 1.1.1.110; the 2,6-dihydroxy-hexanoate CoA-transferase is an enzyme having an EC number 2.8.3, EC number 2.8.3.1, or EC number 2.8.3.12; the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase is an enzyme having an EC number 4.2.1.167; the 2,3-dehydro-hexanoyl-CoA 2,3-reductase is an enzyme having an EC number 1.3.1.44; the 6-hydroxyhexanoyl-CoA transferase is an enzyme having an EC number 2.8.3, EC number 2.8.3.1, or EC number 2.8.3.12; the 6-hydroxyhexanoate dehydrogenase is an enzyme having an EC number 1.1.1.258; and the 6-oxo-hexanoate oxidase is an enzyme having an EC number 1.2.1.63.

In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase is an enzyme selected from the group of enzymes identified under Uniprot or GenBank ID Nos. WP_003431407.1, BAL51292.1, Q5FTU6, AKC64094.1, WP_002876862.1, AGP69017.1, WP_003640741.1, AKC6409; the 2,6-dihydroxy-hexanoate CoA-transferase is an enzyme identified under Uniprot ID Nos. T4VW93 or A0A2X3BTQ9; the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase is an enzyme identified under Uniprot ID Nos. Q5U924, Q5U925, and Q5U923; or A0A2X3BK09, A0A2X3BU19, and A0A1V9IXA9; the 2,3-dehydro-hexanoyl-CoA 2,3-reductase is an enzyme identified under Uniprot ID No. Q73Q47; the 6-hydroxyhexanoyl-CoA transferase is an enzyme identified under Uniprot ID Nos. T4VW93 or A0A2X3BTQ9; the 6-hydroxyhexanoate dehydrogenase is an enzyme identified under Uniprot ID Nos. Q7WVD0 or Q84H78; and the 6-oxo-hexanoate oxidase is an enzyme identified under Uniprot ID No. Q9R2F4.

In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme identified under Uniprot or GenBank ID Nos. WP_003431407.1, BAL51292.1, Q5FTU6, AKC64094.1, WP_002876862.1, AGP69017.1, WP_003640741.1, AKC64095.1, and AKC64094.1; the 2,6-dihydroxy-hexanoate CoA-transferase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme identified under Uniprot ID Nos. T4VW93 or A0A2X3BTQ9; the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme identified under Uniprot ID Nos. Q5U924, Q5U925, and Q5U923; or A0A2X3BK09, A0A2X3BU19, and A0A1V9IXA9; the 2,3-dehydro-hexanoyl-CoA 2,3-reductase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme identified under Uniprot ID No. Q73Q47; the 6-hydroxyhexanoyl-CoA transferase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme identified under Uniprot ID Nos. T4VW93 or A0A2X3BTQ9; the 6-hydroxyhexanoate dehydrogenase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme identified under Uniprot ID Nos. Q7WVD0 or Q84H78; and the 6-oxo-hexanoate oxidase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme identified under Uniprot ID No. Q9R2F4.

In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase is an enzyme comprising a sequence of SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, or SEQ ID NO:105; the 2,6-dihydroxy-hexanoate CoA-transferase is an enzyme comprising a sequence of SEQ ID NO:55 or SEQ ID NO:58; the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase is an enzyme comprising a sequence of SEQ ID NO:59, SEQ ID NO:61, and SEQ ID NO:63; or SEQ ID NO:60, SEQ ID NO:62, and SEQ ID NO:64; the 2,3-dehydro-hexanoyl-CoA 2,3-reductase is an enzyme comprising a sequence of SEQ ID NO:65; the 6-hydroxyhexanoyl-CoA transferase is an enzyme comprising a sequence of SEQ ID NO:55 or SEQ ID NO:58; the 6-hydroxyhexanoate dehydrogenase is an enzyme identified comprising a sequence of SEQ ID NO:71 or SEQ ID NO:72; and the 6-oxo-hexanoate oxidase is an enzyme comprising a sequence of SEQ ID NO:75.

In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme comprising a sequence of SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, or SEQ ID NO:105; the 2,6-dihydroxy-hexanoate CoA-transferase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme comprising a sequence of SEQ ID NO:55 or SEQ ID NO:58; the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme comprising a sequence of SEQ ID NO:59, SEQ ID NO:61, and SEQ ID NO:63; or SEQ ID NO:60, SEQ ID NO:62, and SEQ ID NO:64; the 2,3-dehydro-hexanoyl-CoA 2,3-reductase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme comprising a sequence of SEQ ID NO:65; the 6-hydroxyhexanoyl-CoA transferase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme comprising a sequence of SEQ ID NO:55 or SEQ ID NO:58; the 6-hydroxyhexanoate dehydrogenase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme identified comprising a sequence of SEQ ID NO:71 and SEQ ID NO:72; and the 6-oxo-hexanoate oxidase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme comprising a sequence of SEQ ID NO:75.

In some embodiments, wherein one or more of the 6-hydroxy-2-oxohexanoate-2-reductase, the 2,6-dihydroxy-hexanoate CoA-transferase, the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase, the 2,3-dehydro-hexanoyl-CoA 2,3-reductase, the 6-hydroxyhexanoyl-CoA transferase, 6-hydroxyhexanoate dehydrogenase, and the 6-oxo-hexanoate oxidase are further comprise one or more protein tags. In some embodiments, the protein tags are selected from polyhistidine tag, a GST tag (glutathione-S-transferase tag), a HA tag (hemagglutinin tag), a FLAG tag, a Myc tag, a maltose binding protein tag, a chitin binding protein tag, and a fluorescent tag.

In some embodiments, the method for producing a adipic acid further comprises or consists essentially of separating the adipic acid from the one or more non-naturally occurring microbial organisms or a culture comprising the one or more non-naturally occurring microbial organisms. In some embodiments, the method further comprises or consists essentially of separating the adipic acid from the two or more non-naturally occurring microbial organisms or a culture comprising the two or more non-naturally occurring microbial organisms.

In some embodiments, the pyruvate is produced from carbon sources is selected from glycerol, glucose, xylose, arabinose, galactose, mannose, fructose, sucrose, and starch, or a combination thereof. In some embodiments,

is 3-hydroxy-propanal. In some embodiments, the 3-hydroxy-propanal is produced by dehydration of glycerol by a glycerol dehydratase enzyme exogenously expressed by the one or more non-naturally occurring microbial organisms.

In another aspect, provided herein is a recombinant microbial organism comprising a first exogenous nucleic acid encoding an aldolase hydratase enzyme, wherein the recombinant microbial organism is further modified to express an increased amount of quinone oxidoreductase as compared to wild-type or the same microbial organism that is not modified, and optionally wherein the microbial organism is Corynebacterium glutamicum, a clostridium species, or E. coli. In some embodiments, the organism comprises a second exogenous nucleic acid encoding quinone oxidoreductase. In some embodiments, the first and/or second exogenous nucleic acid further comprises a regulatory element that drives expression of the second exogenous nucleic acid. Alternatively, the first and second nucleic are under the control of the same promoter regulatory element. In some embodiments, the regulatory element is selected from a promoter or an enhancer. In some embodiments, the aldolase hydratase enzyme has an EC number 4.1.2.45 or EC number 4.1.2.34 or EC number 4.1.1.4. In some embodiments, the aldolase hydratase enzyme is an enzyme selected from the group of enzymes identified under Uniprot ID Nos. D7C0E5, P0A144, Q79EM8, A0A0N0AHI8, A0A0N1FRY3, M3DYR1, W7SU48, A0A286PH18, Q9X9Q6, Q9WXH7, A4XDS1, F2J6N9, A0A063BFL5, Q9ZHH6, A0A0C1K853, WP_034398482, PYK12191, WP_115478033, WP_028222253, WP_013654807, WP_059403060, WP_092508530, WP_116642627, WP_009770659, WP_107818191, WP_003292061, PYN48855, WP_122212965, WP_028217297, WP_034507049, KMK64081.1, WP_070028041.1, or KZL92449.1. In some embodiments, the aldolase hydratase enzyme is an enzyme selected from the group of enzymes identified under Uniprot ID Nos. D7C0E5, P0A144, Q79EM8, A0A0N0AHI8, A0A0N1FRY3, M3DYR1, W7SU48, A0A286PH18, Q9X9Q6, Q9WXH7, A4XDS1, F2J6N9, A0A063BFL5, Q9ZHH6, A0A0C1K853, WP_034398482, PYK12191, A0A370X7D8, WP_028222253, F2J6L6, A0A0N0L9F6, A0A1G9YWG7, A0A2U1BT09, A0A244DHE8, WP_107818191, A0A023WZF9, PYN48855, A0A421PAQ6, WP_028217297, WP_034507049, KMK64081.1, WP_070028041.1, or KZL92449.1. In some embodiments, the aldolase hydratase enzyme is an enzyme comprising a sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:84, SEQ ID NO:85, or SEQ ID NO:86.

In some embodiments, the first exogenous nucleic acid and the second exogenous nucleic acid are each contained in a vector, e.g., a plasmid or viral vector. In some embodiments, the first exogenous nucleic acid and the second exogenous nucleic acid are each contained in the same vector. In some embodiments, the first exogenous nucleic acid and the second exogenous nucleic acid are each contained in their own separate vectors. In some embodiments, the vector is a plasmid. In some embodiments, a quinone oxidoreductase is an enzyme having an EC number 1.6.5. In some embodiments, a quinone oxidoreductase is an enzyme having an EC number 1.6.5.5. In some embodiments, the quinone oxidoreductase is an enzyme selected from the group of enzymes identified under GenBank, RefSeq, or Uniprot ID Nos. P28304, P40783, Q0K2I0, A0A1Z1SRY9, P43903, 17G8G0, or Q142L2, ALK19324.1, A0A1G9R408, G4Q8R5, ANA98723.1, K0EUQ3, A0A061CRS8, Q9A212, A0A1I6RWW2, WP_026197277.1, Q5NKZ3, WP_012333034.1, or WP_136898000.1. In some embodiments, the quinone oxidoreductase is an enzyme comprising a sequence of SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, or SEQ ID NO:97. In some embodiments, the recombinant microbial organism is capable of producing a 2-keto carboxylic acid of formula:

wherein R is H, CH₃, or CH₂OH. In some embodiments, the recombinant microbial organism is capable of producing 1,5-pentanediol, 1,6-hexanediol, adipic acid, or 6-hydroxy hexanoate. In some embodiments, the recombinant microbial organism is genetically modified to improve production of pyruvate from a carbon source. In some embodiments, the carbon source is selected from glycerol, glucose, xylose, arabinose, galactose, mannose, fructose, sucrose, and starch, or a combination thereof.

In another aspect, provided herein is a culture comprising the recombinant microbial organisms disclosed herein.

In another aspect, provided herein is a population of recombinant microbial organisms as disclosed herein. In some embodiments, the population is substantially homogenous.

In another aspect, provided herein is a culture comprising the populations disclosed herein.

In another aspect, provided herein is a method of producing 1,5-pentanediol, 1,6-hexanediol, adipic acid, or 6-hydroxy hexanoate, comprising culturing the population or recombinant microorganisms as disclosed herein under suitable conditions that promote expression of the exogenous nucleic acids as disclosed herein. In one aspect, the exogenous nucleic acids are overexpressed as compared to a wild-type or unmodified counterpart microbial organism. In some embodiments, the method further comprises isolating the 1,5-pentanediol, 1,6-hexanediol, adipic acid, or 6-hydroxy hexanoate from the culture or the microbial organisms.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a two-enzyme biosynthetic pathway for production of 2-keto carboxylic acids from pyruvate and aldehydes as an example. An aldol-dehydration product (e.g., an aldol condensation product described herein) can be generated from a process catalyzed by a single enzyme (e.g., an aldol-dehydration product biosynthesis polypeptide such as a hydratase-aldolase (in some embodiments, referred as Ads-Hyd) through, without the intention to be limited by theory, step 1 and 2 as depicted. As those skilled in the art will appreciate, the double bond in the illustrated aldol condensation product may exist as E or Z. In many embodiments, step 3 as illustrated can catalyzed by an oxidoreductase, e.g., one belonging to EC 1.6.5 (e.g., EC 1.6.5.5) that utilizes NADH and/or NADPH for reduction of quinones. As described herein, various aldehydes may be utilized. For example, in the illustrated aldehydes in some embodiments, R is H, CH₃, CH₂CH₃, OH, CH₂OH, or CH₂CH₂OH.

FIG. 2 shows a biosynthetic pathway for production of 1,5-pentanediol via 6-hydroxy-2-keto-hexanoate (6H2KH) intermediate. As used herein 3HPA refers to 3-hydroxy-propanal; 6H4H2KH refers to 4,6-dihydroxy-2-keto-hexanoate; 6H3(E)2KH refers to 6-hydroxy-3,4-dehydro-2-keto-hexenoate; and 5HPeA refers to 5-hydroxy pentanal. NADH is depicted as the cofactors for many reduction steps of the pathway for illustrative purposes. Either NADPH or NADH could be a cofactor.

FIG. 3 shows a biosynthetic pathway for production of 1,6-hexanediol via 6-hydroxy-2-keto-hexanoate (6H2KH) intermediate. As used herein 3HPA refers to 3-hydroxy-propanal; 6H4H2KH refers to 4,6-dihydroxy-2-keto-hexanoate; 6H3(E)2KH refers to 6-hydroxy-3,4-dehydro-2-keto-hexenoate; 6H2HH refers to 2,6-dihydroxy-hexanoate; 6-HH-CoA refers to 6-hydroxy-hexanoyl-CoA; 6HH refers to 6-hydroxy hexanoate; 6H2-HH-CoA refers to 2,6-dihydroxy-hexanoyl-CoA; and 6-HHA refers to 6-hydroxy hexanal. Either NADPH or NADH could be a cofactor. Step 5 and 8 are catalyzed by a single CoA-transferase enzyme. 6HH-CoA is depicted as donor for Step 5 reaction and 6H2HH as the acceptor for illustrative purposes. Other CoA-esters or carboxylic acids can serve as donors and acceptors for this enzyme in vivo.

FIG. 4 shows a biosynthetic pathway for production of 6-hydroxy hexanoate via 6-hydroxy-2-keto-hexanoate (6H2KH) intermediate. As used herein 3HPA refers to 3-hydroxy-propanal; 6H4H2KH refers to 4,6-dihydroxy-2-keto-hexanoate; 6H3(E)2KH refers to 6-hydroxy-3,4-dehydro-2-keto-hexenoate; 6H2-HH refers to 2,6-dihydroxy-hexanoate; 6-HH-CoA refers to 6-hydroxy-hexanoyl-CoA; 6HH refers to 6-hydroxy hexanoate; and 6H2HH-CoA refers to 2,6-dihydroxy-hexanoyl-CoA. Either NADPH or NADH could be a cofactor. Step 5 and 8 are catalyzed by a single CoA-transferase enzyme. 6-HH-CoA is depicted as donor for Step 5 reaction and 6H2-HH as the acceptor for illustrative purposes. Other CoA-esters or carboxylic acids can serve as donors and acceptors for this enzyme in vivo.

FIG. 5 shows biosynthetic pathway for production of adipic acid via 6-hydroxy-2-keto-hexanoate (6H2KH) intermediate. As used herein 3HPA refers to 3-hydroxy-propanal; 6H4H2KH refers to 4,6-dihydroxy-2-keto-hexanoate; 6H3(E)2KH refers to 6-hydroxy-3,4-dehydro-2-keto-hexenoate; 6H2-HH refers to 2,6-dihydroxy-hexanoate; 6HH-CoA refers to 6-hydroxy-hexanoyl-CoA; 6HH refers to 6-hydroxy hexanoate; 6H2-HH-CoA refers to 2,6-dihydroxy-hexanoyl-CoA; and 6KHA refers to 6-oxo-hexanoate. Either NADPH or NADH could be a cofactor. Step 5 and 8 are catalyzed by a single CoA-transferase enzyme. 6HH-CoA is depicted as donor for Step 5 reaction and 6H2-HH as the acceptor for illustrative purposes. Other CoA-esters or carboxylic acids can serve as donors and acceptors for this enzyme in vivo.

FIG. 6 shows the activity of the quinone oxidoreductase-1 (Qor-1) for reducing 6-hydroxy-3,4-dehydro-2-keto-hexenoate to 6-hydroxy-2-keto-hexenoate with cofactor NADH and NADPH.

DETAILED DESCRIPTION Definitions

As used herein, certain terms may have the following defined meanings. As used herein, the singular form “a,” “an” and “the” include singular and plural references unless the context clearly indicates otherwise.

As used herein, the term “comprising” is intended to mean that the compositions and methods include the recited elements, but not excluding others. “Consisting essentially of” when used to define compositions and methods, shall mean excluding other elements of any essential significance to the composition or method. “Consisting of” shall mean excluding more than trace elements of other ingredients for claimed compositions and substantial method steps. Aspects defined by each of these transition terms are within the scope of the present disclosure. Accordingly, it is intended that the methods and compositions can include additional steps and components (comprising) or alternatively including steps and compositions of no significance (consisting essentially of) or alternatively, intending only the stated method steps or compositions (consisting of).

As used therein, the term “aldol-dehydration product biosynthesis polypeptide” refers to a polypeptide that is involved in the synthesis of an aldol-dehydration product as described herein. In some embodiments, an aldol-dehydration product biosynthesis polypeptide may be or comprise an aldolase polypeptide, a hydratase, a hydratase-aldolase polypeptide (e.g., a hydratase-aldolase) as described herein. In some embodiments, an aldol-dehydration product biosynthesis polypeptide may be or comprise a hydratase-aldolase polypeptide (e.g., a hydratase-aldolase) as described herein. In some embodiments, an aldol-dehydration product biosynthesis polypeptide has an amino acid sequence that is found in nature, for example in a microbe (e.g., in a reference aldol-dehydration biosynthesis polypeptide found in nature). Alternatively or additionally, in some embodiments, an aldol-dehydration biosynthesis polypeptide shares a characteristic sequence element and/or an overall percent identity with an appropriate reference aldol-dehydration biosynthesis polypeptide (e.g., as is found in nature and/or is presented herein (e.g., in one or more of relevant Tables (e.g., Tables 1 and 5-8))) or a portion thereof (e.g., a portion (e.g., a domain (e.g., a relevant catalytic domain) and/or a set of amino acid residues (which can be continuous or separated)) that promotes a relevant reaction).

As used herein, an “aldol-dehydration product” refers to a compound comprising an aldehyde or ketone group and a double bond conjugated with the aldehyde or ketone group. In some embodiments, an aldol-dehydration product is a compound of formula P-2 or a salt thereof.

As used herein, the term “aldol product” refers to a compound which comprises an aldehyde or ketone group and a hydroxyl group attached to a beta-carbon of an aldehyde or ketone carbonyl group. In some embodiments, an aldol product is a product of an aldol reaction. In some embodiments, an aldol product has a structure formula P-1 or a salt thereof.

As used herein, the term “aldol product biosynthesis polypeptide” refers to a polypeptide that is involved in the synthesis of an aldol product as described herein. In some embodiments, an aldol product biosynthesis polypeptide may be or comprise an aldolase polypeptide, a hydratase-aldolase polypeptide (e.g., a hydratase-aldolase) as described herein. In some embodiments, an aldol product biosynthesis polypeptide is or comprises a aldolase polypeptide as described herein. In some embodiments, an aldol product biosynthesis polypeptide has an amino acid sequence that is found in nature, for example in a microbe (e.g., in a reference aldol biosynthesis polypeptide found in nature). Alternatively or additionally, in some embodiments, an aldol biosynthesis polypeptide shares a characteristic sequence element and/or an overall percent identity with an appropriate reference aldol biosynthesis polypeptide (e.g., as is found in nature and/or is presented herein (e.g., in one or more of relevant Tables)) or a portion thereof (e.g., a portion (e.g., a domain (e.g., a relevant catalytic domain) and/or a set of amino acid residues (which can be continuous or separated)) that promotes a relevant reaction).

As used herein, the term “alkene reduction product biosynthesis polypeptide” refers to a polypeptide that is involved in the conversion of a double bond into a single bond as described herein (and forming an alkene reduction product). In some embodiments, an alkene reduction product biosynthesis polypeptide may be or comprise quinone oxidoreductase as described herein. In some embodiments, an alkene reduction product biosynthesis polypeptide has an amino acid sequence that is found in nature, for example in a microbe (e.g., in a reference alkene reduction biosynthesis polypeptide found in nature). Alternatively or additionally, in some embodiments, an aldol biosynthesis polypeptide shares a characteristic sequence element and/or an overall percent identity with an appropriate reference aldol biosynthesis polypeptide (e.g., as is found in nature and/or is presented herein (e.g., in one or more of relevant Tables)) or a portion thereof (e.g., a portion (e.g., a domain (e.g., a relevant catalytic domain) and/or a set of amino acid residues (which can be continuous or separated)) that promotes a relevant reaction).

As used herein, the term “aliphatic” means a straight-chain (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is completely saturated or that contains one or more units of unsaturation, or a substituted or unsubstituted monocyclic, bicyclic, or polycyclic hydrocarbon ring that is completely saturated or that contains one or more units of unsaturation (but not aromatic), or combinations thereof. In some embodiments, aliphatic groups contain 1-50 aliphatic carbon atoms. In some embodiments, aliphatic groups contain 1-20 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-10 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-9 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-8 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-7 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-6 aliphatic carbon atoms. In still other embodiments, aliphatic groups contain 1-5 aliphatic carbon atoms, and in yet other embodiments, aliphatic groups contain 1, 2, 3, or 4 aliphatic carbon atoms. Suitable aliphatic groups include, but are not limited to, linear or branched, substituted or unsubstituted alkyl, alkenyl, alkynyl groups and hybrids thereof such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl.

As used herein, the term “alkyl” is given its ordinary meaning in the art and may include saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups. In some embodiments, alkyl has 1-100 carbon atoms. In certain embodiments, a straight chain or branched chain alkyl has about 1-20 carbon atoms in its backbone (e.g., C₁-C₂₀ for straight chain, C₂-C20 for branched chain), and alternatively, about 1-10. In some embodiments, cycloalkyl rings have from about 3-10 carbon atoms in their ring structure where such rings are monocyclic, bicyclic, or polycyclic, and alternatively about 5, 6 or 7 carbons in the ring structure. In some embodiments, an alkyl group may be a lower alkyl group, wherein a lower alkyl group comprises 1-4 carbon atoms (e.g., C₁-C₄ for straight chain lower alkyls).

As used herein, the term “aryl”, used alone or as part of a larger moiety as in “aralkyl,” “aralkoxy,” or “aryloxyalkyl,” refers to monocyclic, bicyclic or polycyclic ring systems having a total of five to thirty ring members, wherein at least one ring in the system is aromatic. In some embodiments, an aryl group is a monocyclic, bicyclic or polycyclic ring system having a total of five to fourteen ring members, wherein at least one ring in the system is aromatic, and wherein each ring in the system contains 3 to 7 ring members. In some embodiments, an aryl group is a biaryl group. The term “aryl” may be used interchangeably with the term “aryl ring.” In certain embodiments of the present disclosure, “aryl” refers to an aromatic ring system which includes, but is not limited to, phenyl, biphenyl, naphthyl, binaphthyl, anthracyl and the like, which may bear one or more substituents. Also included within the scope of the term “aryl,” as it is used herein, is a group in which an aromatic ring is fused to one or more non-aromatic rings, such as indanyl, phthalimidyl, naphthimidyl, phenanthridinyl, or tetrahydronaphthyl, and the like.

As used herein, the term “cycloaliphatic,” “carbocycle,” “carbocyclyl,” “carbocyclic radical,” and “carbocyclic ring,” are used interchangeably, and refer to saturated or partially unsaturated, but non-aromatic, cyclic aliphatic monocyclic, bicyclic, or polycyclic ring systems, as described herein, having, unless otherwise specified, from 3 to 30 ring members. Cycloaliphatic groups include, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, cyclooctyl, cyclooctenyl, norbornyl, adamantyl, and cyclooctadienyl. In some embodiments, a cycloaliphatic group has 3-6 carbons. In some embodiments, a cycloaliphatic group is saturated and is cycloalkyl. The term “cycloaliphatic” may also include aliphatic rings that are fused to one or more aromatic or nonaromatic rings, such as decahydronaphthyl or tetrahydronaphthyl. In some embodiments, a cycloaliphatic group is bicyclic. In some embodiments, a cycloaliphatic group is tricyclic. In some embodiments, a cycloaliphatic group is polycyclic. In some embodiments, “cycloaliphatic” refers to C₃-C₆ monocyclic hydrocarbon, or C₈-C₁₀ bicyclic or polycyclic hydrocarbon, that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic, that has a single point of attachment to the rest of the molecule, or a C₉-C₁₆ polycyclic hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic, that has a single point of attachment to the rest of the molecule.

As used herein, the term “heteroaliphatic” is given its ordinary meaning in the art and refers to aliphatic groups as described herein in which one or more carbon atoms are independently replaced with one or more heteroatoms (e.g., oxygen, nitrogen, sulfur, silicon, phosphorus, and the like). In some embodiments, one or more units selected from C, CH, CH₂, and CH₃ are independently replaced by one or more heteroatoms (including oxidized and/or substituted forms thereof). In some embodiments, a heteroaliphatic group is heteroalkyl. In some embodiments, a heteroaliphatic group is heteroalkenyl.

As used herein, the term “heteroalkyl” is given its ordinary meaning in the art and refers to alkyl groups as described herein in which one or more carbon atoms are independently replaced with one or more heteroatoms (e.g., oxygen, nitrogen, sulfur, silicon, phosphorus, and the like). Examples of heteroalkyl groups include, but are not limited to, alkoxy, poly(ethylene glycol)-, alkyl-substituted amino, tetrahydrofuranyl, piperidinyl, morpholinyl, etc.

As used herein, the terms “heteroaryl” and “heteroar-”, used alone or as part of a larger moiety, e.g., “heteroaralkyl,” or “heteroaralkoxy,” refer to monocyclic, bicyclic or polycyclic ring systems having a total of five to thirty ring members, wherein at least one ring in the system is aromatic and at least one aromatic ring atom is a heteroatom. In some embodiments, a heteroaryl group is a group having 5 to 10 ring atoms (i.e., monocyclic, bicyclic or polycyclic), in some embodiments 5, 6, 9, or 10 ring atoms. In some embodiments, a heteroaryl group has 6, 10, or 14 π electrons shared in a cyclic array; and having, in addition to carbon atoms, from one to five heteroatoms. Heteroaryl groups include, without limitation, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, and pteridinyl. In some embodiments, a heteroaryl is a heterobiaryl group, such as bipyridyl and the like. The terms “heteroaryl” and “heteroar-”, as used herein, also include groups in which a heteroaromatic ring is fused to one or more aryl, cycloaliphatic, or heterocyclyl rings, where the radical or point of attachment is on the heteroaromatic ring. Non-limiting examples include indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and pyrido[2,3-b]-1,4-oxazin-3(4H)-one. A heteroaryl group may be monocyclic, bicyclic or polycyclic. The term “heteroaryl” may be used interchangeably with the terms “heteroaryl ring,” “heteroaryl group,” or “heteroaromatic,” any of which terms include rings that are optionally substituted. The term “heteroaralkyl” refers to an alkyl group substituted by a heteroaryl group, wherein the alkyl and heteroaryl portions independently are optionally substituted.

As used herein, the term “heteroatom” refers to an atom that is not carbon or hydrogen. In some embodiments, a heteroatom is boron, oxygen, sulfur, nitrogen, phosphorus, or silicon (including oxidized forms of nitrogen, sulfur, phosphorus, or silicon; charged forms of nitrogen (e.g., quaternized forms, forms as in iminium groups, etc.), phosphorus, sulfur, oxygen; etc.). In some embodiments, a heteroatom is oxygen, sulfur or nitrogen.

As used herein, the terms “heterocycle,” “heterocyclyl,” “heterocyclic radical,” and “heterocyclic ring”, as used herein, are used interchangeably and refer to a monocyclic, bicyclic or polycyclic ring moiety (e.g., 3-30 membered) that is saturated or partially unsaturated and has one or more heteroatom ring atoms. In some embodiments, a heterocyclyl group is a stable 5- to 7-membered monocyclic or 7- to 10-membered bicyclic heterocyclic moiety that is either saturated or partially unsaturated, and having, in addition to carbon atoms, one or more, preferably one to four, heteroatoms, as defined above. When used in reference to a ring atom of a heterocycle, the term “nitrogen” includes substituted nitrogen. As an example, in a saturated or partially unsaturated ring having 0-3 heteroatoms selected from oxygen, sulfur and nitrogen, the nitrogen may be N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl), or ⁺NR (as in N-substituted pyrrolidinyl). A heterocyclic ring can be attached to its pendant group at any heteroatom or carbon atom that results in a stable structure and any of the ring atoms can be optionally substituted. Examples of such saturated or partially unsaturated heterocyclic radicals include, without limitation, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and quinuclidinyl. The terms “heterocycle,” “heterocyclyl,” “heterocyclyl ring,” “heterocyclic group,” “heterocyclic moiety,” and “heterocyclic radical,” are used interchangeably herein, and also include groups in which a heterocyclyl ring is fused to one or more aryl, heteroaryl, or cycloaliphatic rings, such as indolinyl, 3H-indolyl, chromanyl, phenanthridinyl, or tetrahydroquinolinyl. A heterocyclyl group may be monocyclic, bicyclic or polycyclic. The term “heterocyclylalkyl” refers to an alkyl group substituted by a heterocyclyl, wherein the alkyl and heterocyclyl portions independently are optionally substituted.

Optionally Substituted: As described herein, chemical entities, e.g., various compounds, of the disclosure may contain optionally substituted and/or substituted moieties. In general, the term “substituted” means that one or more hydrogens of the designated moiety are replaced with a suitable substituent. Unless otherwise indicated, an “optionally substituted” group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position. In some embodiments, an optionally substituted group is substituted. In some embodiments, an optionally substituted group is unsubstituted. Combinations of substituents envisioned by this disclosure are preferably those that result in the formation of stable or chemically feasible compounds. The term “stable,” as used herein, refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and, in certain embodiments, their recovery, purification, and use for one or more of the purposes disclosed herein. Certain substituents are described below.

Suitable monovalent substituents on a substitutable atom, e.g., a suitable carbon atom, are independently halogen; —(CH₂)₀₋₄R^(o); —(CH₂)₀₋₄OR^(o); —O(CH₂)₀₋₄R^(o), —O—(CH₂)₀₋₄C(O)OR^(o); —(CH₂)₀₋₄ CH(OR^(o))₂; —(CH₂)₀₋₄ Ph, which may be substituted with R^(o); —(CH₂)₀₋₄O(CH₂)₀₋₁Ph which may be substituted with R^(o); —CH═CHPh, which may be substituted with R^(o); —(CH₂)₀₋₄O(CH₂)₀₋₁-pyridyl which may be substituted with R^(o); —NO₂; —CN; —N₃; —(CH₂)₀₋₄N(R^(o))₂; —(CH₂)₀₋₄N(R^(o))C(O)R^(o); —N(R^(o))C(S)R^(o); —(CH₂)₀₋₄N(R^(o))C(O)NR^(o) ₂; —N(R^(o))C(S)NR^(o) ₂; —(CH₂)₀₋₄N(R^(o))C(O)OR^(o); —N(R^(o))N(R^(o))C(O)R^(o); —N(R^(o))N(R^(o))C(O)NR^(o) ₂; —N(R^(o))N(R^(o))C(O)OR^(o); —(CH₂)₀₋₄C(O)R^(o); —C(S)R^(o); —(CH₂)₀₋₄C(O)OR^(o); —(CH₂)₀₋₄C(O)SR^(o); —(CH₂)₀₋₄C(O)OSiR^(o) ₃; —(CH₂)₀₋₄ OC(O)R^(o); —OC(O)(CH₂)₀₋₄ SR^(o), —SC(S)SR^(o); —(CH₂)₀₋₄ SC(O)R^(o); —(CH₂)₀₋₄C(O)NR^(o) ₂; —C(S)NR^(o) ₂; —C(S)SR^(o); —(CH₂)₀₋₄OC(O)NR^(o) ₂; —C(O)N(OR^(o))R^(o); —C(O)C(O)R^(o); —C(O)CH₂C(O)R^(o); —C(NOR^(o))R^(o); —(CH₂)₀₋₄SSR^(o); —(CH₂)₀₋₄S(O)₂R^(o); —(CH₂)₀₋₄S(O)₂OR^(o); —(CH₂)₀₋₄OS(O)₂R^(o); —S(O)₂NR^(o) ₂; —(CH₂)₀₋₄ S(O)R^(o); —N(R^(o))S(O)₂NR^(o) ₂; —N(R^(o))S(O)₂R^(o); —N(OR^(o))R^(o); —C(NH)NR^(o))₂; —Si(R^(o))₃; —OSi(R^(o))₃; —B(R^(o))₂; —OB(R^(o))₂; —OB(OR^(o))₂; —P(R^(o))₂; —P(OR^(o))₂; —P(R^(o))(OR^(o); —OP(R^(o))₂; —OP(OR^(o))₂; —OP(R^(o))(OR^(o); —P(O)(R^(o))₂; —P(O)(OR^(o))₂; —OP(O)(R^(o))₂; —OP(O)(OR^(o))₂; —OP(O)(OR^(o))(SR^(o); —SP(O)(R^(o))₂; —SP(O)(OR^(o))₂; —N(R^(o))P(O)(R^(o))₂; —N(R^(o))P(O)(OR^(o))₂; —P(R^(o))₂[B(R^(o))₃]; —P(OR^(o))₂[B(R^(o))₃]; —OP(R^(o))₂[B(R^(o))₃]; —OP(OR^(o))₂[B(R^(o))₃]; —(C₁₋₄ straight or branched alkylene)O—N(R^(o))₂; or —(C₁₋₄ straight or branched)alkylene)C(O)O—N(R^(o) ₂, wherein each R^(o) may be substituted as defined herein and is independently hydrogen, C₁₋₂₀ aliphatic, C₁₋₂₀ heteroaliphatic having 1-5 heteroatoms independently selected from nitrogen, oxygen, sulfur, silicon and phosphorus, —CH₂—(C₆₋₁₄ aryl), —O(CH₂)₀₋₁ (C₆₋₁₄ aryl), —CH₂-(5-14 membered heteroaryl ring), a 5-20 membered, monocyclic, bicyclic, or polycyclic, saturated, partially unsaturated or aryl ring having 0-5 heteroatoms independently selected from nitrogen, oxygen, sulfur, silicon and phosphorus, or, notwithstanding the definition above, two independent occurrences of R^(o), taken together with their intervening atom(s), form a 5-20 membered, monocyclic, bicyclic, or polycyclic, saturated, partially unsaturated or aryl ring having 0-5 heteroatoms independently selected from nitrogen, oxygen, sulfur, silicon and phosphorus, which may be substituted as defined below.

Suitable monovalent substituents on R^(o) (or the ring formed by taking two independent occurrences of R^(o) together with their intervening atoms), are independently halogen, —(CH₂)₀₋₂R^(●), -(haloR^(●)), —(CH₂)₀₋₂OH, —(CH₂)₀₋₂OR^(●), —(CH₂)₀₋₂ CH(OR^(●))₂; —O(haloR^(●)), —CN, —N₃, —(CH₂)₀₋₂C(O)R^(●), —(CH₂)₀₋₂C(O)OH, —(CH₂)₀₋₂C(O)OR^(●), —(CH₂)₀₋₂ SR^(●), —(CH₂)₀₋₂ SH, —(CH₂)₀₋₂NH₂, —(CH₂)₀₋₂NHR^(●), —(CH₂)₀₋₂NR^(●) ₂, —NO₂, —OSiR^(●) ₃, —C(O)SR^(●), —(C₁₋₄ straight or branched alkylene)C(O)OR^(●), or —SSR^(●) wherein each R^(●) is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently selected from C₁₋₄ aliphatic, —CH₂Ph, —O(CH₂)₀₋₁Ph, and a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. Suitable divalent substituents on a saturated carbon atom of R^(o) include ═O and ═S.

Suitable divalent substituents, e.g., on a suitable carbon atom, are independently the following: ═O, ═S, ═NNR^(*) ₂, ═NNHC(O)R*, ═NNHC(O)OR*, ═NNHS(O)₂R*, ═NR*, ═NOR*, —O(C(R*₂))₂₋₃O—, or —S(C(R*₂))₂₋₃S—, wherein each independent occurrence of R* is selected from hydrogen, C₁₋₆ aliphatic which may be substituted as defined below, and an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. Suitable divalent substituents that are bound to vicinal substitutable carbons of an “optionally substituted” group include: —O(CR*₂)₂₋₃O—, wherein each independent occurrence of R* is selected from hydrogen, C₁₋₆ aliphatic which may be substituted as defined below, and an unsubstituted 5-6-membered saturated, partially unsaturated, and aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

Suitable substituents on the aliphatic group of R* are independently halogen, —R^(●), -(haloR^(●)), —OH, —OR^(●), —O(haloR^(●)), —CN, —C(O)OH, —C(O)OR^(●), —NH₂, —NHR^(●), —NR^(●) ₂, or —NO₂, wherein each R^(●) is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently C₁₋₄ aliphatic, —CH₂Ph, —O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

In some embodiments, suitable substituents on a substitutable nitrogen are independently —R^(†), —NR^(†) ₂, —C(O)R^(†), —C(O)OR^(†), —C(O)C(O)R^(†), —C(O)CH₂C(O)R^(†), —S(O)₂R^(†), —S(O)₂NR^(†) ₂, —C(S)NR^(†) ₂, —C(NH)NR^(†) ₂, or —N(R^(†))S(O)₂R^(†); wherein each R^(†) is independently hydrogen, C₁₋₆ aliphatic which may be substituted as defined below, unsubstituted —OPh, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or, notwithstanding the definition above, two independent occurrences of R^(†), taken together with their intervening atom(s) form an unsubstituted 3-12-membered saturated, partially unsaturated, or aryl mono- or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

Suitable substituents on the aliphatic group of R^(†) are independently halogen, —R^(▪), -(haloR^(▪)), —OH, —OR^(▪), —O(haloR^(▪)), —CN, —C(O)OH, —C(O)OR^(▪), —NH₂, —NH^(▪), —NR^(▪) ₂, or —NO₂, wherein each R^(▪) is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently C₁ aliphatic, —CH₂Ph, —O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

As used herein, the term “partially unsaturated” refers to a ring moiety that includes at least one double or triple bond. The term “partially unsaturated” is intended to encompass rings having multiple sites of unsaturation, but is not intended to include aryl or heteroaryl moieties, as herein defined.

“Wild-type” defines the cell, composition, tissue or other biological material as it exists in nature.

In some embodiments, the 3-hydroxy-propanal and pyruvate are prepared from one or more of glycerol, C5 sugars, C6 sugars, phosphor-glycerates, other carbon sources, intermediates of the glycolysis pathway, and combinations thereof. In some embodiments, the C5 sugars comprise or alternatively consists essentially of, or yet further consists of, one or more of xylose, xylulose, ribulose, arabinose, lyxose, and ribose, and the C6 sugars comprise or alternatively consist essentially of, or yet further consist of, allose, altrose, glucose, mannose, gulose, idose, talose, fructose, psicose, sorbose, and tagatose. In some embodiments, the other carbon source is a feedstock suitable as a carbon source for a microorganism wherein the feedstock comprises or alternatively consists essentially of, or yet further consists of, one or more of amino acids, lipids, corn stover, miscanthus, municipal waste, energy cane, sugar cane, bagasse, starch stream, dextrose stream, formate, methanol, and combinations thereof.

As used herein, the term “C5 sugar” refers to a sugar molecule containing 5 carbons.

As used herein, the term “C6 sugar” refers to a sugar molecule containing 6 carbons.

In some embodiments, the term “aldol addition” refers to a chemical reaction in which a pyruvate molecule forms a corresponding enol or an enolate ion or a Schiff's base or an enamine that reacts with the aldehyde functional group of the CN aldehyde to produce a C_(N+3) 4-hydroxy-2-keto-carboxylic acid intermediate. In some embodiments, the CN aldehyde is 3-hydroxy-propanal and the C_(N+3) 4-hydroxy-2-keto-carboxylic acid intermediate is 4,6-dihydroxy-2-keto-hexanoic acid.

In some embodiments, the term “aldol condensation” refers to a chemical reaction in which a pyruvate molecule forms a corresponding enol or an enolate ion or a Schiff's base or an enamine that reacts with the aldehyde functional group of the CN aldehyde to produce a C_(N+3) 3,4-dehydro-2-keto-carboxylic acid. In some embodiments, the C_(N) aldehyde is 3-hydroxy-propanal and the C_(N+3) 3,4-dehydro-2-keto-carboxylic acid is 6-hydroxy-3,4-dehydro-2-keto-hexanoic acid.

As used herein, the term “solution” refers to a liquid composition that contains a solvent and a solute, such as a starting material used in the methods described herein. In some embodiments, the solvent is water. In some embodiments, the solvent is an organic solvent.

As used herein, the term “enzymatic step” or “enzymatic reaction” refers to a molecular reaction catalyzed by an enzyme that is selected to facilitate the desired enzymatic reaction. Enzymes are large biological molecules and highly selective catalysts. Most enzymes are proteins, but some catalytic RNA molecules have been identified.

Throughout the application, enzymatic steps may be denoted as “step 1”, “step 2” and so on so forth and the enzyme specifically catalyzing these steps is denoted as “1”, “2” and so on so forth, respectively. Such an enzyme is also referred to as a “reaction specific enzyme”.

As used herein, the term “CoA” or “coenzyme A” is intended to mean an organic cofactor or prosthetic group (nonprotein portion of an enzyme) whose presence is required for the activity of many enzymes to form an active enzyme system.

As used herein, the term “substantially anaerobic” when used in reference to a culture or growth condition is intended to mean that the amount of oxygen is less than about 10% of saturation for dissolved oxygen in liquid media. The term also is intended to include sealed chambers of liquid or solid medium maintained with an atmosphere of less than about 1% oxygen.

As used herein, the term “non-naturally occurring” or “non-natural” when used in reference to a microbial organism or microorganism of the present disclosure is intended to mean that the microbial organism has at least one genetic alteration not normally found in a naturally occurring strain of the referenced species, including wild-type strains of the referenced species. Genetic alterations include, for example, but are not limited to, modifications introducing expressible nucleic acids encoding polypeptides, other nucleic acid additions, nucleic acid deletions and/or other functional disruption of the microbial organism's genetic material. Such modifications include, for example, but are not limited to, coding regions and functional fragments thereof, for heterologous, homologous or both heterologous and homologous polypeptides for the referenced species. Additional modifications include, for example, but are not limited to, non-coding regulatory regions in which the modifications alter expression of a gene or operon.

As is used herein “exogenous” is intended to mean that the referenced molecule or the referenced activity is introduced into the host microbial organism. The molecule can be introduced, for example, by introduction of an encoding nucleic acid into the host genetic material such as by integration into a host chromosome or as non-chromosomal genetic material such as a plasmid. Therefore, the term as it is used in reference to expression of an encoding nucleic acid refers to introduction of the encoding nucleic acid in an expressible form into the microbial organism. When used in reference to an enzymatic activity, the term refers to an activity that is introduced into the host reference organism. The source can be, for example, a homologous or heterologous encoding nucleic acid that expresses the referenced activity following introduction into the host microbial organism. Therefore, the term “endogenous” refers to a referenced molecule or activity that is originally or naturally present in the wild-type host. Similarly, the term when used in reference to expression of an encoding nucleic acid refers to expression of an encoding nucleic acid contained within the wild-type microorganism.

The term “heterologous” refers to a molecule or activity derived from a source other than the referenced species whereas “homologous” when used in this context refers to a molecule or activity derived from the host microbial organism. Accordingly, exogenous expression of an encoding nucleic acid can utilize either or both a heterologous or homologous encoding nucleic acid.

It is understood that when more than one exogenous nucleic acid is included in a microbial organism, that the more than one exogenous nucleic acids refers to the referenced encoding nucleic acid or enzymatic activity, as discussed above. It is further understood, as disclosed herein, that more than one exogenous nucleic acids can be introduced into the host microbial organism on separate nucleic acid molecules, on polycistronic nucleic acid molecules, or a combination thereof, and still be considered as more than one exogenous nucleic acid. For example, as disclosed herein, a microbial organism can be engineered to express two or more exogenous nucleic acids encoding a desired pathway enzyme or protein. In the case where two exogenous nucleic acids encoding a desired activity are introduced into a host microbial organism, it is understood that the two exogenous nucleic acids can be introduced as a single nucleic acid, for example, on a single plasmid, on separate plasmids, can be integrated into the host chromosome at a single site or multiple sites, and still be considered as two exogenous nucleic acids. Similarly, it is understood that more than two exogenous nucleic acids can be introduced into a host organism in any desired combination, for example, on a single plasmid, on separate plasmids, can be integrated into the host chromosome at a single site or multiple sites, and still be considered as two or more exogenous nucleic acids, for example three exogenous nucleic acids. Thus, the number of referenced exogenous nucleic acids or enzymatic activities refers to the number of encoding nucleic acids or the number of enzymatic activities, not the number of separate nucleic acids introduced into the host organism.

In some embodiments, exogenous expression of the encoding nucleic acids is employed. Exogenous expression confers the ability to custom tailor the expression and/or regulatory elements to the host and application to achieve a desired expression level that is controlled by the user. However, endogenous expression also can be utilized in other embodiments such as by removing a negative regulatory effector or induction of the gene's promoter when linked to an inducible promoter or other regulatory element. Thus, an endogenous gene having a naturally occurring inducible promoter can be up-regulated by providing the appropriate inducing agent, or the regulatory region of an endogenous gene can be engineered to incorporate an inducible regulatory element, thereby allowing the regulation of increased expression of an endogenous gene at a desired time. Similarly, an inducible promoter can be included as a regulatory element for an exogenous gene introduced into a non-naturally occurring microbial organism.

Those skilled in the art will understand that the genetic alterations are described with reference to a suitable host organism such as E. coli and their corresponding metabolic reactions or a suitable source organism for desired genetic material such as genes for a desired biosynthetic pathway. However, given the complete genome sequencing of a wide variety of organisms and the high level of skill in the area of genomics, those skilled in the art will readily be able to apply the teachings and guidance provided herein to essentially all other organisms. For example, the E. coli metabolic alterations exemplified herein can readily be applied to other species by incorporating the same or analogous encoding nucleic acid from species other than the referenced species. Such genetic alterations include, for example, genetic alterations of species homologs, in general, and in particular, orthologs, paralogs or nonorthologous gene displacements.

Sources of encoding nucleic acids the pathway enzymes can include, for example, any species where the encoded gene product is capable of catalyzing the referenced reaction. Such species include both prokaryotic and eukaryotic organisms including, but not limited to, bacteria, including archaea and eubacteria, and eukaryotes, including yeast, plant, insect, animal, and mammal, including human. Exemplary species for such sources include, for example, Escherichia coli, Pseudomonas knackmussii, Pseudomonas putida, Pseudomonas fluorescens, Klebsiella pneumoniae, Serratia proteamaculans, Streptomyces sp. 2065, Pseudomonas aeruginosa, Ralstonia eutropha, Clostridium acetobutylicum, Euglena gracilis, Treponema denticola, Clostridium kluyveri, Homo sapiens, Rattus norvegicus, Acinetobacter sp. ADP1, Streptomyces coelicolor, Eubacterium barkeri, Peptostreptococcus asaccharolyticus, Clostridium botulinum, Clostridium tyrobutyricum, Clostridium thermoaceticum (Moorella thermoaceticum), Acinetobacter calcoaceticus, Mus musculus, Sus scrofa, Flavobacterium sp, Arthrobacter aurescens, Penicillium chrysogenum, Aspergillus niger, Aspergillus nidulans, Bacillus subtilis, Saccharomyces cerevisiae, Zymomonas mobilis, Mannheimia succiniciproducens, Clostridium ljungdahlii, Clostridium carboxydivorans, Geobacillus stearothermophilus, Agrobacterium tumefaciens, Achromobacter denitrificans, Arabidopsis thaliana, Haemophilus influenzae, Acidaminococcus fermentans, Clostridium sp. M62/1, Fusobacterium nucleatum, as well as other exemplary species disclosed herein or available as source organisms for corresponding genes (see Examples). However, with the complete genome sequence available for now more than 400 microorganism genomes and a variety of yeast, fungi, plant, and mammalian genomes, the identification of genes encoding the requisite pathway enzymes, for one or more genes in related or distant species, including for example, homologues, orthologs, paralogs and nonorthologous gene displacements of known genes, and the interchange of genetic alterations between organisms is routine and well known in the art.

Ortholog refers to genes in different species that evolved from a common ancestral gene by speciation. Normally, orthologs retain the same function in the course of evolution. Identification of orthologs is critical for reliable prediction of gene function in newly sequenced genomes.

Paralog refers to genes related by duplication within a genome. While orthologs generally retain the same function in the course of evolution, paralogs can evolve new functions, even if these are related to the original one.

A nonorthologous gene displacement is a nonorthologous gene from one species that can substitute for a referenced gene function in a different species. Substitution includes, for example, being able to perform substantially the same or a similar function in the species of origin compared to the referenced function in the different species. Although generally, a nonorthologous gene displacement will be identifiable as structurally related to a known gene encoding the referenced function, less structurally related but functionally similar genes and their corresponding gene products nevertheless will still fall within the meaning of the term as it is used herein. Functional similarity requires, for example, at least some structural similarity in the active site or binding region of a nonorthologous gene product compared to a gene encoding the function sought to be substituted. Therefore, a nonorthologous gene includes, for example, a paralog or an unrelated gene.

As used herein, the terms “microorganism” or “microbial organism” or “microbes” are used interchangeably and refer to a living biological and isolated prokaryotic or eukaryotic cell that can be transformed or transfected via insertion of an exogenous or recombinant nucleic acid, such as DNA or RNA. Any suitable prokaryotic or eukaryotic microorganism may be used in the present disclosure so long as it remains viable after being transformed with a sequence of nucleic acids. A suitable microorganism of the present disclosure is one capable of expressing one or more nucleic acid constructs encoding one or more recombinant proteins that can catalyze at least one step in the methods. Microorganism can be selected from group of bacteria, yeast, fungi, mold, and archaea. These are commercially available.

As used herein, “fungal” refers to any eukaryotic organism categorized within the kingdom of Fungi. Phyla within the kingdom of Fungi include Ascomycota, Basidiomycota, Blastocladiomycota, Chytridiomycota, Glomeromycota, Microsporidia, and Neocallimastigomycota. As used herein, “yeast” refers to fungi growing in single-celled forms (for example, by budding), whereas “mold” refers to fungi growing in filaments made of multicellular hyphae or mycelia (McGinnis, M. R. and Tyring, S. K. “Introduction to Mycology.” Medical Microbiology. 4^(th) ed. Galveston: Univ. of TX Medical Branch at Galveston, 1996).

In some embodiments, the microorganisms are yeast cells. In some embodiments, the yeast cell is from a Candida, Hansenula, Issatchenkia, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia species.

In some embodiments, the microorganisms are mold cells. In some embodiments, the mold host cell is from a Neurospora, Trichoderma, Aspergillus, Fusarium, or Chrysosporium species.

In some embodiments, the microorganism is an archaea. In some embodiments, a suitable archaea is from an Archaeoglobus, Aeropyrum, Halobacterium, Pyrobaculum, Pyrococcus, Sulfolobus, Methanococcus, Methanosphaera, Methanopyrus, Methanobrevibacter, Methanocaldococcus, or Methanosarcina species.

The term “bacteria” refers to any microorganism within the domain or kingdom of prokaryotic organisms. Phyla within the domain or kingdom of bacteria include Acidobacteria, Actinobacteria, Actinobacillus, Agrobacterium, Anaerobiospirrulum, Aquificae, Armatimonadetes, Bacteroidetes, Burkholderia, Caldiserica, Chlamydiae, Chlorobi, Chlorella, Chloroflexi, Chrysiogenetes, Citrobacter, Clostridium, Cyanobacteria, Deferribacteres, Deinococcus-thermus, Dictyoglomi, Enterobacter, Elusimicrobia, Fibrobacteres, Firmicutes, Fusobacteria, Geobacillus, Gemmatimonadetes, Gluconobacter, Halanaerobium, Klebsiella, Kluyvera, Lactobacillus, Lentisphaerae, Methylobacterium, Nitrospira, Pasteurellaceae, Paenibacillus, Planctomycetes, Propionibacterium, Pseudomonas, Proteobacteria, Ralstonia, Schizochytrium, Spirochaetes, Streptomyces, Synergistetes, Tenericutes, Thermoanaerobacterium, Thermodesulfobacteria, Thermotogae, Verrucomicrobia, Zobellella, and Zymomonas. In some embodiments, the bacterial microorganisms are E. coli cells. In some embodiments, the bacterial microorganisms are Bacillus sp. cells. Examples of Bacillus species include without limitation Bacillus subtilis, Bacillus megaterium, Bacillus cereus, Bacillus thuringiensis, Bacillus mycoides, and Bacillus licheniformis.

A carboxylic acid compound prepared by the methods of the present disclosure can form a salt with a counter ion including, but not limited to, a metal ion, e.g., an alkali metal ion, such as sodium, potassium, an alkaline earth ion, such as calcium, magnesium, or an aluminum ion; or coordinates with an organic base such as tetraalkylammonium, ethanolamine, diethanolamine, triethanolamine, trimethylamine, N-methylglucamine, and the like. The acid can form a salt with a counter ion or organic base present in the reaction conditions or can be converted to a salt by reacting with an inorganic or organic base.

Any carboxylic acid containing compound herein is referred to as either an acid or a salt, which has been used interchangeably throughout to refer to the compound in any of its neutral or ionized forms, including any salt forms thereof. It is understood by those skilled understand that the specific form will depend on the pH.

A solvate of a compound is a solid-form of the compound that crystallizes with less than one, one or more than one molecules of solvent inside in the crystal lattice. A few examples of solvents that can be used to create solvates, such as pharmaceutically acceptable solvates, include, but are not limited to, water, C₁-C₆ alcohols (such as methanol, ethanol, isopropanol, butanol, and can be optionally substituted) in general, tetrahydrofuran, acetone, ethylene glycol, propylene glycol, acetic acid, formic acid, and solvent mixtures thereof. Other such biocompatible solvents which may aid in making a pharmaceutically acceptable solvate are well known in the art. Additionally, various organic and inorganic acids and bases can be added to create a desired solvate. Such acids and bases are known in the art. When the solvent is water, the solvate can be referred to as a hydrate. In some embodiments, one molecule of a compound can form a solvate with from 0.1 to 5 molecules of a solvent, such as 0.5 molecules of a solvent (hemisolvate, such as hemihydrate), one molecule of a solvent (monosolvate, such as monohydrate) and 2 molecules of a solvent (disolvate, such as dihydrate).

When referring to a compound for which several isomers exist (e.g., cis and trans isomer, and R and S isomer, or a combination thereof), the compound in principle includes all possible enantiomers, diastereomers and cis/trans isomers of that compound that may be used in the method of the present disclosure.

For each species, any cell belonging to that species is considered a suitable microorganism of the present disclosure. A host cell of any species may exist as it was isolated from nature, or it may contain any number of genetic modifications (e.g., genetic mutations, deletions, or recombinant polynucleotides).

The term “recombinant nucleic acid” or “recombinant polynucleotide” as used herein refers to a polymer of nucleic acids where at least one of the following is true: (a) the sequence of nucleic acids is foreign to (i.e., not naturally found in) a given microorganism; (b) the sequence may be naturally found in a given microorganism, but in an unnatural (e.g., greater than expected) amount; or (c) the sequence of nucleic acids contains two or more subsequences that are not found in the same relationship to each other in nature. For example, regarding instance (c), a recombinant nucleic acid sequence will have two or more sequences from unrelated genes arranged to make a new functional nucleic acid.

In some embodiments, recombinant polypeptides or proteins or enzymes of the present disclosure may be encoded by genetic material as part of one or more expression vectors. An expression vector contains one or more polypeptide-encoding nucleic acids, and it may further contain any desired elements that control the expression of the nucleic acid(s), as well as any elements that enable the replication and maintenance of the expression vector inside a given host cell. All of the recombinant nucleic acids may be present on a single expression vector, or they may be encoded by multiple expression vectors.

An expression vector or vectors can be constructed to include one or more pathway-encoding nucleic acids as exemplified herein operably linked to expression control sequences functional in the host organism. Expression vectors applicable for use in the microbial host organisms provided include, for example, plasmids, phage vectors, viral vectors, episomes and artificial chromosomes, including vectors and selection sequences or markers operable for stable integration into a host chromosome. Additionally, the expression vectors can include one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes also can be included that, for example, provide resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply critical nutrients not in the culture media. Expression control sequences can include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like which are well known in the art. When two or more exogenous encoding nucleic acids are to be co-expressed, both nucleic acids can be inserted, for example, into a single expression vector or in separate expression vectors. For single vector expression, the encoding nucleic acids can be operationally linked to one common expression control sequence or linked to different expression control sequences, such as one inducible promoter and one constitutive promoter. Vectors that contain both a promoter and a cloning site into which a polynucleotide can be operatively linked are well known in the art. Such vectors are capable of transcribing RNA in vitro or in vivo, and are commercially available from sources such as Stratagene (La Jolla, Calif.) and Promega Biotech (Madison, Wis.). In order to optimize expression and/or in vitro transcription, it may be necessary to remove, add or alter 5′ and/or 3′ untranslated portions of the clones to eliminate extra, potential inappropriate alternative translation initiation codons or other sequences that may interfere with or reduce expression, either at the level of transcription or translation. Alternatively, consensus ribosome binding sites can be inserted immediately 5′ of the start codon to enhance expression.

Exogenous nucleic acid sequences involved in a pathway for synthesis of desired compounds described herein can be introduced stably or transiently into a host cell using techniques well known in the art including, but not limited to, conjugation, electroporation, chemical transformation, transduction, transfection, and ultrasound transformation. For exogenous expression in E. coli or other prokaryotic cells, some nucleic acid sequences in the genes or cDNAs of eukaryotic nucleic acids can encode targeting signals such as an N-terminal mitochondrial or other targeting signal, which can be removed before transformation into prokaryotic host cells, if desired. For example, removal of a mitochondrial leader sequence led to increased expression in E. coli (Hoffmeister et al., J. Biol. Chem. 280:4329-4338 (2005)). For exogenous expression in yeast or other eukaryotic cells, genes can be expressed in the cytosol without the addition of leader sequence, or can be targeted to mitochondrion or other organelles, or targeted for secretion, by the addition of a suitable targeting sequence such as a mitochondrial targeting or secretion signal suitable for the host cells. It is understood that appropriate modifications to a nucleic acid sequence to remove or include a targeting sequence can be incorporated into an exogenous nucleic acid sequence to impart desirable properties. Furthermore, genes can be subjected to codon optimization with techniques well known in the art to achieve optimized expression of the proteins.

All numerical designations, e.g., pH, temperature, time, concentration, and molecular weight, including ranges, are approximations which are varied (+) or (−) by increments of 0.1. It is to be understood, although not always explicitly stated that all numerical designations are preceded by the term “about”. As used herein, “about” will mean up to plus or minus 10%. It also is to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art.

“Operatively linked” refers to a juxtaposition wherein the elements are in an arrangement allowing them to function.

The term “culturing” refers to the in vitro propagation of cells or organisms on or in media (culture) of various kinds. It is understood that the descendants of a cell grown in culture may not be completely identical (i.e., morphologically, genetically, or phenotypically) to the parent cell.

A “gene” refers to a polynucleotide containing at least one open reading frame (ORF) that is capable of encoding a particular polypeptide or protein after being transcribed and translated. Any of the polynucleotide sequences described herein may be used to identify larger fragments or full-length coding sequences of the gene with which they are associated. Methods of isolating larger fragment sequences are known to those of skill in the art.

The term “express” refers to the production of a gene product. The term overexpression refers to the production of the mRNA transcribed from the gene or the protein product encoded by the gene that is more than that of a normal or control cell, for example 0.5 times, 1.0 times, 1.5 times, or alternatively, 2 times, or alternatively, at least 2.5 times, or alternatively, at least 3.0 times, or alternatively, at least 3.5 times, or alternatively, at least 4.0 times, or alternatively, at least 5 times, or alternatively 10 times higher than the expression level detected in a control sample or wild-type cell.

As used herein, “homology” refers to sequence similarity between a reference sequence and at least a fragment of a second sequence. Homologs may be identified by any method known in the art, preferably, by using the BLAST tool to compare a reference sequence to a single second sequence or fragment of a sequence or to a database of sequences. As described below, BLAST will compare sequences based upon percent identity and similarity.

The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same. Two sequences are “substantially identical” if two sequences have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 29% identity, optionally 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identity over a specified region, or, when not specified, over the entire sequence), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Optionally, the identity exists over a region that is at least about 50 nucleotides (or 10 amino acids) in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides (or 20, 50, 200, or more amino acids) in length.

Methods of alignment of sequences for comparison are well-known in the art. For example, the determination of percent sequence identity between any two sequences can be accomplished using a mathematical algorithm. Non-limiting examples of such mathematical algorithms are the algorithm of Myers and Miller, CABIOS 4:11 17 (1988); the local homology algorithm of Smith et al., Adv. Appl. Math. 2:482 (1981); the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 453 (1970); the search-for-similarity-method of Pearson and Lipman, Proc. Natl. Acad. Sci. 85:2444 2448 (1988); the algorithm Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873 5877 (1993).

For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. When comparing two sequences for identity, it is not necessary that the sequences be contiguous, but any gap would carry with it a penalty that would reduce the overall percent identity. For blastn, the default parameters are Gap opening penalty=5 and Gap extension penalty=2. For blastp, the default parameters are Gap opening penalty=11 and Gap extension penalty=1.

A “comparison window,” as used herein, includes reference to a segment of any one of the number of contiguous positions including, but not limited to from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman (1981), by the homology alignment algorithm of Needleman and Wunsch, J Mol Biol 48(3):443-453 (1970), by the search for similarity method of Pearson and Lipman, Proc Natl Acad Sci USA 85(8):2444-2448 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection [see, e.g., Brent et al., (2003) Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (Ringbou Ed)].

Two examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nucleic Acids Res 25(17):3389-3402 (1997) and Altschul et al., J. Mol Biol 215(3)-403-410 (1990), respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) or 10, M=5, N=−4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc Natl Acad Sci USA 89(22):10915-10919 (1992)) alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparison of both strands.

The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc Natl Acad Sci USA 90(12):5873-5877 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

Other than percentage of sequence identity noted above, another indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross-reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions. Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequence.

The phrase “functionally equivalent protein” refers to protein or polynucleotide which hybridizes to the exemplified polynucleotide under stringent conditions and which exhibit similar or enhanced biological activity in vivo, e.g., over 120%, or alternatively over 110%, or alternatively over 100%, or alternatively, over 90% or alternatively over 85% or alternatively over 80%, as compared to the standard or control biological activity. Additional embodiments within the scope of the present disclosure are identified by having more than 80%, or alternatively, more than 85%, or alternatively, more than 90%, or alternatively, more than 95%, or alternatively more than 97%, or alternatively, more than 98 or 99% sequence homology. Percentage homology can be determined by sequence comparison programs such as BLAST run under appropriate conditions. In some embodiments, the program is run under default parameters. In some embodiments, reference to a certain enzyme or protein includes its functionally equivalent enzyme or protein.

A population of cells intends a collection of more than one cell that is identical (clonal) or non-identical in phenotype and/or genotype. A substantially homogenous population of cells is a population having at least 70%, or alternatively at least 75%, or alternatively at least 80%, or alternatively at least 85%, or alternatively at least 90%, or alternatively at least 95%, or alternatively at least 98% identical phenotype, as measured by pre-selected markers.

When an enzyme is mentioned with reference to an enzyme class (EC), the enzyme class is a class wherein the enzyme is classified or may be on classified on the basis of the enzyme nomenclature provided by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology. Other suitable enzymes that have not yet been classified in a specific class but may be classified as such are also included.

Non-Naturally Occurring Microbial Organisms

The non-naturally occurring microbial organisms provided herein are constructed using methods well known in the art as exemplified herein to exogenously express at least one nucleic acid encoding an enzyme or protein used in a biosynthetic pathway described herein in sufficient amounts to produce compounds such as 2-keto pentanoic acid, 2-keto hexanoic acid, 6-hydroxy-2-keto-hexanoic acid, 1,5-pentanediol, adipic acid, 1, 6-hexanediol, or 6-hydroxy hexanoic acid.

Successful engineering of a microbial host capable of producing the desired product described herein involves identifying the appropriate set of enzymes with sufficient activity and specificity for catalyzing various steps in the pathway, for example those described in the Examples herein and in literature. The individual enzyme or protein activities from the exogenous DNA sequences can also be assayed using methods well known in the art. In addition, these enzymes can be engineered using modern protein engineering approaches (Protein Engineering Handbook; Lutz S., & Bornscheuer U. T. Wiley-VCH Verlag GmbH & Co. KGaA: 2008; Vol. 1 & 2) such as directed evolution, rational mutagenesis, computational design (Zanghellini, A et al, 2008) or a combination thereof, for achieving the desired substrate specificity, controlling the stereoselectivity to synthesize enantiopure or racemic products, stabilizing the enzyme to withstand harsh industrial process conditions by improving half-life, thermostability, inhibitor/product tolerance and improving enzyme expression and solubility in the desired microbial production host of choice. Once the desired enzymes that can catalyze each step of the pathway are characterized, the genes encoding these enzymes will be cloned in the microorganism of choice, fermentation conditions will be optimized and product formation will be monitored following fermentation. After the enzymes are identified, the genes corresponding to one or more of the enzymes are cloned into a microbial host. In some embodiments, the genes encoding each enzyme of a particular pathway described herein are cloned into a microbial host.

Methods to introduce recombinant/exogenous nucleic acids/proteins into a microorganism, and vectors suitable for this purpose, are well known in the art. For example, various techniques are illustrated in Current Protocols in Molecular Biology, Ausubel et al., eds. (Wiley & Sons, New York, 1988, and quarterly updates). Methods for transferring expression vectors into microbial host cells are well known in the art. Specific methods and vectors may differ depending upon the species of the desired microbial host. For example, bacterial host cells may be transformed by heat shock, calcium chloride treatment, electroporation, liposomes, or phage infection. Yeast host cells may be transformed by lithium acetate treatment (may further include carrier DNA and PEG treatment) or electroporation. These methods are included for illustrative purposes and are in no way intended to be limiting or comprehensive. Routine experimentation through means well known in the art may be used to determine whether a particular expression vector or transformation method is suited for a given microbial host. Furthermore, reagents and vectors suitable for many different microbial hosts are commercially available and well known in the art.

Methods for construction, expression or overexpression of enzymes and testing the expression levels in non-naturally occurring microbial hosts are well known in art (Protein Expression Technologies: Current Status and Future Trends, Baneyx F. eds. Horizon Bioscience, 2004, Norfolk, UK; and Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York (2001); and Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1999)).

Methods for carrying out fermentation of microorganisms are well known in art. For example, various techniques are illustrated in Biochemical Engineering, Clark et al., eds. (CRC press, 1997, 2^(nd) edition). Specific methods for fermenting may differ depending upon the species of the desired microbial host. Typically, the microorganism is grown in appropriate media along with the carbon source in a batch or a continuous fermentation mode. The use of agents known to modulate catabolite repression or enzyme activity can be used to enhance adipic acid or glutaric acid production. Suitable pH for fermentation is between 3-10. Fermentation can be performed under aerobic, anaerobic, or anoxic conditions based on the requirements of the microorganism. Fermentations can be performed in a batch, fed-batch or continuous manner. Fermentations can also be conducted in two phases, if desired. For example, the first phase can be aerobic to allow for high growth and therefore high productivity, followed by an anaerobic phase of high caprolactone yields.

The carbon source can include, for example, any carbohydrate source which can supply a source of carbon to the non-naturally occurring microorganism. Such sources include, for example, sugars such as glucose, xylose, arabinose, galactose, mannose, fructose, sucrose and starch. Other sources of carbohydrate include, for example, renewable feedstocks and biomass. Exemplary types of biomasses that can be used as feedstocks in the methods of the present disclosure include cellulosic biomass, hemicellulosic biomass and lignin feedstocks or portions of feedstocks. Such biomass feedstocks contain, for example, carbohydrate substrates useful as carbon sources such as glucose, xylose, arabinose, galactose, mannose, fructose and starch. Given the teachings and guidance provided herein, those skilled in the art will understand that renewable feedstocks and biomass other than those exemplified above also can be used for culturing the microbial organisms of the present disclosure for the production of desired compound.

The reactions described herein can be monitored and the starting materials, the products or intermediates in the fermentation media can be identified by analyzing the media using high pressure liquid chromatography (HPLC) analysis, GC-MS (Gas Chromatography-Mass Spectroscopy) and LC-MS (Liquid Chromatography-Mass Spectroscopy) or other suitable analytical methods using routine procedures well known in the art.

Any of the non-naturally occurring microbial organisms described herein can be cultured to produce and/or secrete the products of the present disclosure.

Compounds prepared by the methods described herein can be isolated by methods generally known in the art for isolation of an organic compound prepared by biosynthesis or fermentation. For example, the compounds can be isolated from solution by crystallization, salt formation, pervaporation, reactive extraction, extraction (liquid-liquid and two-phase), adsorption, ion exchange, dialysis, distillation, gas stripping, and membrane based separations (Roffler et al., Trends Biotechnolgy. 2: 129-136 (1984)). 1,5-Pentanediol can be isolated from solution using distillation, extraction (liquid-liquid and two-phase), pervaporation, and membrane based separations (Roffler et al., Trends Biotechnolgy. 2: 129-136 (1984)).

As described herein, one exemplary growth condition for achieving biosynthesis of desired product includes anaerobic culture or fermentation conditions. In certain embodiments, the non-naturally occurring microbial organisms of the present disclosure can be sustained, cultured or fermented under anaerobic or substantially anaerobic conditions. Briefly, anaerobic conditions refer to an environment devoid of oxygen. Substantially anaerobic conditions include, for example, a culture, batch fermentation or continuous fermentation such that the dissolved oxygen concentration in the medium remains between 0 and 10% of saturation. Substantially anaerobic conditions also include growing or resting cells in liquid medium or on solid agar inside a sealed chamber maintained with an atmosphere of less than 1% oxygen. The percent of oxygen can be maintained by, for example, sparging the culture with an N₂/CO₂ mixture or other suitable non-oxygen gas or gases.

The culture conditions described herein can be scaled up and grown continuously for manufacturing of products. Exemplary growth procedures include, for example, fed-batch fermentation and batch separation; fed-batch fermentation and continuous separation, or continuous fermentation and continuous separation. All of these processes are well known in the art. Fermentation procedures are particularly useful for the biosynthetic production in commercial quantities.

The term “pathway enzyme expressed in a sufficient amount” implies that the enzyme is expressed in an amount that is sufficient to allow detection of the desired pathway product.

In another aspect, provided herein is a recombinant microbial organism comprising a first exogenous nucleic acid encoding an aldolase hydratase enzyme, wherein the recombinant microbial organism is further modified to express an increased amount of quinone oxidoreductase as compared to wild-type or the same microbial organism that is not modified, and optionally wherein the microbial organism is Corynebacterium glutamicum, a clostridium species, or E. coli.

In some embodiments, the organism comprises a second exogenous nucleic acid encoding quinone oxidoreductase. In some embodiments, the first exogenous nucleic acid and/or the second exogenous nucleic acid further comprises a regulatory element that drives expression of the second exogenous nucleic acid. In some embodiments, the first exogenous nucleic acid and the second exogenous nucleic acid further comprises a regulatory element that drives expression of the second exogenous nucleic acid. In some embodiments, the first exogenous nucleic acid or the second exogenous nucleic acid further comprises a regulatory element that drives expression of the second exogenous nucleic acid. In some embodiments, the first exogenous nucleic acid further comprises a regulatory element that drives expression of the second exogenous nucleic acid. In some embodiments, the second exogenous nucleic acid further comprises a regulatory element that drives expression of the second exogenous nucleic acid. In some embodiments, the regulatory element is selected from a promoter or an enhancer. In some embodiments, the regulatory element is a promoter. In some embodiments, the regulatory element is an enhancer.

In some embodiments, the aldolase hydratase enzyme has an EC number 4.1.2.45, EC number 4.1.2.34 or EC number 4.1.1.4. In some embodiments, the aldolase hydratase enzyme is an enzyme selected from the group of enzymes identified under Uniprot ID Nos. D7C0E5, P0A144, Q79EM8, A0A0N0AHI8, A0A0N1FRY3, M3DYR1, W7SU48, A0A286PH18, Q9X9Q6, Q9WXH7, A4XDS1, F2J6N9, A0A063BFL5, Q9ZHH6, A0A0C1K853, WP_034398482, PYK12191, WP_115478033, WP_028222253, WP_013654807, WP_059403060, WP_092508530, WP_116642627, WP_009770659, WP_107818191, WP_003292061, PYN48855, WP_122212965, WP_028217297, WP_034507049, KMK64081.1, WP_070028041.1, or KZL92449.1. In some embodiments, the hydratase-aldolase is an enzyme selected from the group of enzymes identified under GenBank, RefSeq, or Uniprot ID Nos. D7C0E5, P0A144, Q79EM8, A0A0N0AHI8, A0A0N1FRY3, M3DYR1, W7SU48, A0A286PH18, Q9X9Q6, Q9WXH7, A4XDS1, F2J6N9, A0A063BFL5, Q9ZHH6, A0A0C1K853, WP_034398482, PYK12191, A0A370X7D8, WP_028222253, F2J6L6, A0A0N0L9F6, A0A1G9YWG7, A0A2U1BT09, A0A244DHE8, WP_107818191, A0A023WZF9, PYN48855, A0A421PAQ6, WP_028217297, WP_034507049, KMK64081.1, WP_070028041.1, or KZL92449.1. In some embodiments, the hydratase-aldolase is an enzyme comprising a sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:84, SEQ ID NO:85, or SEQ ID NO:86.

In some embodiments, the hydratase-aldolase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme selected from the group of enzymes identified under GenBank, RefSeq, or Uniprot ID Nos. D7C0E5, P0A144, Q79EM8, A0A0N0AHI8, A0A0N1FRY3, M3DYR1, W7SU48, A0A286PH18, Q9X9Q6, Q9WXH7, A4XDS1, F2J6N9, A0A063BFL5, Q9ZHH6, A0A0C1K853, WP_034398482, PYK12191, A0A370X7D8, WP_028222253, F2J6L6, A0A0N0L9F6, A0A1G9YWG7, A0A2U1BT09, A0A244DHE8, WP_107818191, A0A023WZF9, PYN48855, A0A421PAQ6, WP_028217297, WP_034507049, KMK64081.1, WP_070028041.1, or KZL92449.1, or a portion (e.g., a domain, a set of amino acid residues (can be continuous or separated), etc.) thereof that promotes the formation of a aldol-dehydration product. In some embodiments, the hydratase-aldolase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme comprising a sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:84, SEQ ID NO:85, or SEQ ID NO:86.

In some embodiments, the hydratase-aldolase is an enzyme selected from Tables 1, 5, 6, 7, and 8. In some embodiments, the hydratase-aldolase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme selected from Tables 1, 5, 6, 7, and 8.

In some embodiments, the hydratase-aldolase further comprises one or more protein tags. In some embodiments, the protein tags are selected from polyhistidine tag, a GST tag (glutathione-S-transferase tag), a HA tag (hemagglutinin tag), a FLAG tag, a Myc tag, a maltose binding protein tag, a chitin binding protein tag, and a fluorescent tag.

In some embodiments, the first exogenous nucleic acid and the second exogenous nucleic acid are each contained in a vector. In some embodiments, the first exogenous nucleic acid and the second exogenous nucleic acid are each contained in the same vector. In some embodiments, the first exogenous nucleic acid and the second exogenous nucleic acid are each contained in their own separate vectors. In some embodiments, the vector is a plasmid. In some embodiments, the vector is a viral vector.

In some embodiments, the quinone oxidoreductase is an enzyme having an EC number 1.6.5. In some embodiments, the quinone oxidoreductase is an enzyme having an EC number 1.6.5.5. In some embodiments, the quinone oxidoreductase is an enzyme selected from the group of enzymes identified under GenBank, RefSeq, or Uniprot ID Nos. P28304, P40783, Q0K2I0, A0A1Z1SRY9, P43903, I7G8G0, or Q142L2, ALK19324.1, A0A1G9R408, G4Q8R5, ANA98723.1, K0EUQ3, A0A061CRS8, Q9A212, A0A1I6RWW2, WP_026197277.1, Q5NKZ3, WP_012333034.1, or WP_136898000.1. In some embodiments, the quinone oxidoreductase is an enzyme comprising a sequence of SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, or SEQ ID NO:97.

In some embodiments, the quinone oxidoreductase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme selected from the group of enzymes identified under under GenBank, RefSeq, or Uniprot ID Nos. P28304, P40783, Q0K2I0, A0A1Z1SRY9, P43903, I7G8G0, or Q142L2, ALK19324.1, A0A1G9R408, G4Q8R5, ANA98723.1, K0EUQ3, A0A061CRS8, Q9A212, A0A1I6RWW2, WP_026197277.1, Q5NKZ3, WP_012333034.1, or WP_136898000.1. In some embodiments, the quinone oxidoreductase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme comprising a sequence of SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, or SEQ ID NO:97.

In some embodiments, the quinone oxidoreductase further comprises one or more protein tags. In some embodiments, the protein tags are selected from polyhistidine tag, a GST tag (glutathione-S-transferase tag), a HA tag (hemagglutinin tag), a FLAG tag, a Myc tag, a maltose binding protein tag, a chitin binding protein tag, and a fluorescent tag.

In some embodiments, the recombinant microbial organism is capable of producing a 2-keto carboxylic acid of formula:

wherein R is H, CH₃, or CH₂OH.

In some embodiments, the recombinant microbial organism is capable of producing 1,5-pentanediol, 1,6-hexanediol, adipic acid, or 6-hydroxy hexanoate.

In some embodiments, the recombinant microbial organism is genetically modified to improve production of pyruvate from a carbon source. In some embodiments, the carbon source is selected from glycerol, glucose, xylose, arabinose, galactose, mannose, fructose, sucrose, and starch, or a combination thereof.

In another aspect, provided herein is a population of recombinant microbial organisms disclosed herein. In some embodiments, the population is substantially homogenous. In some embodiments, substantially homogenous refers to at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or more, homogenous.

In another aspect, provided herein is a method of producing 1,5-pentanediol, 1,6-hexanediol, adipic acid, or 6-hydroxy hexanoate, comprising culturing the population disclosed herein under suitable conditions. In some embodiments, the method further comprises isolating the 1,5-pentanediol, 1,6-hexanediol, adipic acid, or 6-hydroxy hexanoate from the culture or the microbial organisms.

Detailed Description of Certain Embodiments

Among other things, the present disclosure encompasses the recognition that certain polypeptides, e.g., various aldol-dehydration product biosynthesis polypeptides which are or comprise hydratase-aldolase polypeptides, can be utilized to effectively produce various compounds. In some embodiments, the present disclosure demonstrates that various aldehydes, e.g., various aliphatic aldehydes described herein, which are structurally different from natural and/or known aldehyde substrates of such polypeptides, can be utilized for effective manufacturing of many products using aldol-dehydration product biosynthesis polypeptide described herein. Among other things, the present disclosure demonstrates that production of various aldol-dehydration products can be catalyzed by a single aldol-dehydration product biosynthesis polypeptide (e.g., various hydratase-aldolase polypeptides as described herein).

In some embodiments, the present disclosure provides a method comprising:

contacting pyruvate and an aldehyde with an aldol-dehydration product biosynthesis polypeptide so that an aldol-dehydration product is produced, wherein:

the aldol-dehydration product is a compound comprising an aldehyde or ketone group and a double bond conjugated with the aldehyde or ketone group.

In some embodiments, an aldehyde is an aliphatic aldehyde. In some embodiments, a —CHO group of an aldehyde is not conjugated, e.g., to a double bond, a triple bond or an aromatic group.

In some embodiments, the present disclosure provides a method comprising:

contacting pyruvate and an aliphatic aldehyde with an aldol-dehydration product biosynthesis polypeptide so that an aldol-dehydration product is produced, wherein:

the carbonyl group of the aliphatic aldehyde is not conjugated to a alkenyl, alkynyl, or aromatic group; and

the aldol-dehydration product is a compound comprising an aldehyde or ketone group and a double bond conjugated with the aldehyde or ketone group.

In some embodiments, an aldol-dehydration product biosynthesis polypeptide is or comprises a hydratase-aldolase polypeptide, e.g., those exemplified herein. In some embodiments, provided methods comprise contacting pyruvate and an aliphatic aldehyde with a hydratase-aldolase to produce an aldol-dehydration product.

In some embodiments, an aldol-dehydration product biosynthesis polypeptide comprises an aldolase polypeptide. In some embodiments, an aldol-dehydration product biosynthesis polypeptide comprises a hydratase polypeptide. In some embodiments, an aldol-dehydration product biosynthesis polypeptide comprises a hydratase-aldolase polypeptide. In some embodiments, an aldol-dehydration product biosynthesis polypeptide is a hydratase-aldolase polypeptide. In some embodiments, a hydratase-aldolase polypeptide is or comprises a hydratase-aldolase as described herein, e.g., an enzyme having an EC number 4.1.2.45 or EC number 4.1.2.34, or EC 4.1.1.4, or is selected from Tables 1 and 5-8.

In some embodiments, an aldol-dehydration product biosynthesis polypeptide is within an organism, e.g., a microbe. In some embodiments, an organism expresses an engineered aldol-dehydration product biosynthesis polypeptide. In some embodiments, an organism expresses an increased level and/or activity of aldol-dehydration product biosynthesis polypeptide. In some embodiments, an organism provides an increased rate and/or yield for producing an aldol-dehydration product. In some embodiments, an organism provides an increased substrate utilization for producing an aldol-dehydration product.

In some embodiments, conversion of pyruvate and an aliphatic aldehyde into an aldol-dehydration product is catalyzed by an aldol-dehydration product biosynthesis polypeptide.

In some embodiments, an aldol-dehydration product can be provided through alternative pathways. In some embodiments, an aldol-dehydration product is produced from an aldol product.

In some embodiments, the present disclosure provides a method comprising:

contacting pyruvate and an aldehyde with an aldol product biosynthesis polypeptide so that an aldol product is produced, wherein:

the aldol-dehydration product is a compound comprising an aldehyde or ketone group and a double bond conjugated with the aldehyde or ketone group.

In some embodiments, an aldehyde is an aliphatic aldehyde. In some embodiments, a —CHO group of an aldehyde is not conjugated to a double bond, triple bond or an aromatic group.

In some embodiments, the present disclosure provides a method comprising:

contacting pyruvate and an aliphatic aldehyde with an aldol product biosynthesis polypeptide so that an aldol product is produced, wherein:

the carbonyl group of the aliphatic aldehyde is not conjugated to a alkenyl, alkynyl, or aromatic group; and

the aldol product is a compound comprising an aldehyde or ketone group and a hydroxyl group attached to a beta-carbon of an aldehyde or ketone carbonyl group.

Various methods of the present disclosure comprise utilization of biosynthesis polypeptides. In some embodiments, a biosynthesis polypeptide, when used together with a particular product, e.g., an aldol product biosynthesis polypeptide, a reduction product biosynthesis polypeptide, etc., refers to a polypeptide that is involved in the synthesis of the particular product. In some embodiments, a biosynthesis polypeptide when used together with a particular product is or comprises an enzyme that catalyzes formation of the particular product. In some embodiments, a biosynthesis polypeptide has an amino acid sequence that is found in nature, for example in a microbe (e.g., in a reference biosynthesis polypeptide for a particular product found in nature). Alternatively or additionally, in some embodiments, a biosynthesis polypeptide shares a characteristic sequence element and/or an overall percent identity with an appropriate reference biosynthesis polypeptide (e.g., as is found in nature and/or is presented herein (e.g., in one or more of relevant Tables) or a portion thereof (e.g., a portion (e.g., a domain (e.g., a relevant catalytic domain) and/or a set of amino acid residues (which can be continuous or separated)) that promotes a relevant reaction).

In some embodiments, an aldol product biosynthesis polypeptide is or comprises an aldolase polypeptide. Those skilled in the art reading the present disclosure appreciate that various aldolase polypeptides can be utilized in accordance with the present disclosure. In some embodiments, an aldolase polypeptide is or comprises an aldolase described in US20170044551, the aldolases of which are incorporated herein by reference.

In some embodiments, an aldol product biosynthesis polypeptide is or comprises an aldolase-hydratase as described herein.

In some embodiments, an aldol product biosynthesis polypeptide is in an organism such as a microbe. In some embodiments, organisms are engineered to express an engineered or exogenous aldol product biosynthesis polypeptides, often at higher protein levels and/or activity levels. In some embodiments, conversion of pyruvate and an aliphatic aldehyde into an aldol product is catalyzed by an aldol product biosynthesis polypeptide. In some embodiments, a method is performed in a culture, e.g., a bacteria culture. As for other biosynthesis polypeptides, aldol product biosynthesis polypeptides may be in organisms such as bacteria, may be engineered, and/or may be expressed at increased at increased protein and/or activity levels, and their products may be generated at increased rates and/or yields and/or substrates utilization.

In some embodiments, an aldol product is converted into an aldol-dehydration product, either catalyzed by an enzyme, through biosynthesis, or through traditional organic synthesis without enzymatic catalysis. In some embodiments, a conversion comprises contacting an aldol product with a dehydration product biosynthesis polypeptide so that an aldol-dehydration product is produced. In some embodiments, a dehydration product biosynthesis polypeptide is or comprises a hydratase. In some embodiments, a dehydration product biosynthesis polypeptide is or comprises a dehydratase. In some embodiments, a hydratase or dehydratase is described in US20170044551, the hydratases and dehydratases of which are incorporated herein by reference. As for other biosynthesis polypeptides, dehydration product biosynthesis polypeptides may be in organisms such as bacteria, may be engineered, and/or may be expressed at increased at increased protein and/or activity levels, and their products may be generated at increased rates and/or yields and/or substrates utilization.

As appreciated by those skilled in the art, aldol-dehydration products can be utilized to manufacture various products, e.g., 1,5-pentanediol, 1,6-hexanediol, 6HH, adipic acid, etc. which can be utilized to manufacture a wide range of products, such as polymers, resins, coating products, etc. In some embodiments, utilization of aldol-dehydration products comprises one or more chemical conversions, each of which may be independently catalyzed by a polypeptide (e.g., an enzyme described herein), optionally in an organism, or performed through traditional chemical processes without utilization of enzymes. As appreciated by those skilled in the art, one or more or all steps can be performed in one or more organisms, each of which may independently perform one or more reactions using substrate(s) generated within itself or from outside of the organism, and/or one or more cultures which independently comprises one or more types of organisms (each of which may independently perform one or more reactions using substrate(s) generated within itself or from a culture (e.g., a feed compound, a compound generated by another organism, etc.)). In some embodiments, one or more or all biosynthesis polypeptides are independently in one organism, e.g., an bacterium optionally engineered. In some embodiments, one or more of a set of biosynthesis polypeptides for producing a product is expressed in one organism, e.g., an bacterium optionally engineered, and one or more of the other biosynthesis polypeptides in the set is expressed in one or more other organisms, e.g., bacteria optionally engineered. In some embodiments, an organism, e.g., a bacterium is engineered to contain one or more exogenous nucleic acids that encode one or more or all of the biosynthesis polypeptides. In some embodiments, manufacturing of a product comprises multiple steps of reactions which are performed in a single culture comprising one or more bacteria each independently comprises one or more or all, and together comprise all, required biosynthesis polypeptides. In some embodiments, manufacturing of a product comprises multiple steps of reactions which are performed in two or more cultures each independently comprising one or more bacteria each independently comprises one or more or all, and together comprise all, required biosynthesis polypeptides.

For example, in some embodiments, double bonds in aldol-dehydration products are converted to single bonds.

In some embodiments, the present disclosure provides a method comprising:

contacting an alkene with an alkene reduction product biosynthesis polypeptide so that an alkene reduction product is produced, wherein:

the alkene comprises a double bond conjugated to a carbonyl group; and

a double bond conjugated to a carbonyl group in the alkene is reduced to a single bond to provide an alkene reduction product.

In some embodiments, an alkene is an aldol-dehydration product.

In some embodiments, an alkene reduction product biosynthesis polypeptide is or comprises an enzyme that catalyze reduction of aldol-dehydration product, e.g., 2-oxo-3-enoic acids, as described herein. In some embodiments, such an enzyme is a quinone oxidoreductase as described herein. In some embodiments, such an enzyme belongs to EC 1.6.5. In some embodiments, such an enzyme belongs to EC 1.6.5.5. In some embodiments, such an enzyme is selected from Table 9.

In some embodiments, alkene reduction product biosynthesis polypeptide is within an organism, e.g., a microbe. In some embodiments, an organism expresses an engineered alkene reduction product biosynthesis polypeptide. In some embodiments, an organism expresses an increased level and/or activity of alkene reduction product biosynthesis polypeptide. In some embodiments, an organism provides an increased rate and/or yield for producing an alkene reduction product. In some embodiments, an organism provides an increased substrate utilization for producing an alkene reduction product.

In some embodiments, an alkene reduction product biosynthesis polypeptide is or comprises an enzyme that encoded and/or expressed by an organism endogenously without engineering.

Those skilled in the art reading the present disclosure appreciate that various aldehydes may be utilized in accordance with the present disclosure. In some embodiments, an aldehyde is a natural or known substrate of a biosynthesis polypeptide, e.g., aldol-dehydration product biosynthesis polypeptide which is or comprises a hydratase-aldolase. In some embodiments, an aldehyde is not a natural or known substrate. For example, among other things, the present disclosure demonstrates that aliphatic aldehydes can be utilized for product manufacturing using hydratase-aldolases whose natural or known substrates are aromatic or conjugated aldehydes.

In some embodiments, an aldehyde is an aliphatic aldehyde. In some embodiments, an aldehyde has one or two alpha-hydrogen. In some embodiments, an aldehyde has the structure of formula A-1:

R^(a)-L²-L¹-C(O)H,   A-1

or a salt thereof, wherein:

R^(a) is R″ or —OR″,

each of L¹ and L² is independently a covalent bond, or a bivalent, optionally substituted, linear or branched C₁₋₂₀ aliphatic or C₁₋₂₀ heteroaliphatic, wherein one or more methylene units are optionally and independently replaced by —C≡C—, —C(R″)₂—, -Cy-, —O—, —S—, —S—S—, —N(R″)—, —C(O)—, —C(S)—, —C(NR″)—, —C(O)N(R″)—, —N(R″)C(O)N(R″)—, —N(R″)C(O)O—, —S(O)—, —S(O)₂—, —S(O)₂N(R″)—, —C(O)S—, or —C(O)O—;

-Cy- is a bivalent, optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring, wherein each monocyclic ring is independently an optionally substituted, saturated, partially saturated or aromatic 3-20 membered ring having 0-5 heteroatoms;

each R″ is independently —R′, —C(O)R′, —CO₂R′, or —SO₂R′;

R′ is hydrogen, or an optionally substituted group selected from C₁₋₁₀ aliphatic, C₁₋₁₀ heteroaliphatic having 1-5 heteroatoms, a 6-10 membered aryl ring, a 5-10 membered heteroaryl ring having 1-5 heteroatoms, and a 3-10 membered heterocyclic ring having 1-5 heteroatoms, or:

two or more R′ groups are taken together with their intervening atoms to form an optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring having, in addition to the intervening atoms, 0-5 heteroatoms, wherein each monocyclic ring is independently an optionally substituted, saturated, partially saturated or aromatic 3-20 membered ring having 0-5 heteroatoms.

In some embodiments, an aldol product has the structure of formula P-1:

R^(a)-L²-L¹-CH(OH)—CH₂—C(O)—C(O)OH,   P-1

or a salt thereof, wherein:

R^(a) is R″ or —OR″,

each of L¹ and L² is independently a covalent bond, or a bivalent, optionally substituted, linear or branched C₁₋₂₀ aliphatic or C₁₋₂₀ heteroaliphatic, wherein one or more methylene units are optionally and independently replaced by —C≡C—, —C(R″)₂—, -Cy-, —O—, —S—, —S—S—, —N(R″)—, —C(O)—, —C(S)—, —C(NR″)—, —C(O)N(R″)—, —N(R″)C(O)N(R″)—, —N(R″)C(O)O—, —S(O)—, —S(O)₂—, —S(O)₂N(R″)—, —C(O)S—, or —C(O)O—;

-Cy- is a bivalent, optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring, wherein each monocyclic ring is independently an optionally substituted, saturated, partially saturated or aromatic 3-20 membered ring having 0-5 heteroatoms;

each R″ is independently —R′, —C(O)R′, —CO₂R′, or —SO₂R′;

R′ is hydrogen, or an optionally substituted group selected from C₁₋₁₀ aliphatic, C₁₋₁₀ heteroaliphatic having 1-5 heteroatoms, a 6-10 membered aryl ring, a 5-10 membered heteroaryl ring having 1-5 heteroatoms, and a 3-10 membered heterocyclic ring having 1-5 heteroatoms, or:

two or more R′ groups are taken together with their intervening atoms to form an optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring having, in addition to the intervening atoms, 0-5 heteroatoms, wherein each monocyclic ring is independently an optionally substituted, saturated, partially saturated or aromatic 3-20 membered ring having 0-5 heteroatoms.

In some embodiments, an aldol-dehydration product has the structure of formula P-2:

R^(a)-L²-L¹-CH═CH—C(O)—C(O)OH,   P-2

or a salt thereof, wherein:

R^(a) is R″ or —OR″,

each of L¹ and L² is independently a covalent bond, or a bivalent, optionally substituted, linear or branched C₁₋₂₀ aliphatic or C₁₋₂₀ heteroaliphatic, wherein one or more methylene units are optionally and independently replaced by —C≡C—, —C(R″)₂—, -Cy-, —O—, —S—, —S—S—, —N(R″)—, —C(O)—, —C(S)—, —C(NR″)—, —C(O)N(R″)—, —N(R″)C(O)N(R″)—, —N(R″)C(O)O—, —S(O)—, —S(O)₂—, —S(O)₂N(R″)—, —C(O)S—, or —C(O)O—;

-Cy- is a bivalent, optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring, wherein each monocyclic ring is independently an optionally substituted, saturated, partially saturated or aromatic 3-20 membered ring having 0-5 heteroatoms;

each R″ is independently —R′, —C(O)R′, —CO₂R′, or —SO₂R′;

R′ is hydrogen, or an optionally substituted group selected from C₁₋₁₀ aliphatic, C₁₋₁₀ heteroaliphatic having 1-5 heteroatoms, a 6-10 membered aryl ring, a 5-10 membered heteroaryl ring having 1-5 heteroatoms, and a 3-10 membered heterocyclic ring having 1-5 heteroatoms, or:

two or more R′ groups are taken together with their intervening atoms to form an optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring having, in addition to the intervening atoms, 0-5 heteroatoms, wherein each monocyclic ring is independently an optionally substituted, saturated, partially saturated or aromatic 3-20 membered ring having 0-5 heteroatoms.

In some embodiments, —CH═CH— in formula P-2 is in E configuration. In some embodiments, —CH═CH— in formula P-2 is in Z configuration.

In some embodiments, an alkene reduction product has the structure of formula P-3:

R^(a)-L²-L¹-CH₂—CH₂—C(O)—C(O)OH,  P-3

or a salt thereof, wherein:

R^(a) is R″ or —OR″,

each of L¹ and L² is independently a covalent bond, or a bivalent, optionally substituted, linear or branched C₁₋₂₀ aliphatic or C₁₋₂₀ heteroaliphatic, wherein one or more methylene units are optionally and independently replaced by —C≡C—, —C(R″)₂—, -Cy-, —O—, —S—, —S—S—, —N(R″)—, —C(O)—, —C(S)—, —C(NR″)—, —C(O)N(R″)—, —N(R″)C(O)N(R″)—, —N(R″)C(O)O—, —S(O)—, —S(O)₂—, —S(O)₂N(R″)—, —C(O)S—, or —C(O)O—;

-Cy- is a bivalent, optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring, wherein each monocyclic ring is independently an optionally substituted, saturated, partially saturated or aromatic 3-20 membered ring having 0-5 heteroatoms;

each R″ is independently —R′, —C(O)R′, —CO₂R′, or —SO₂R′;

R′ is hydrogen, or an optionally substituted group selected from C₁₋₁₀ aliphatic, C₁₋₁₀ heteroaliphatic having 1-5 heteroatoms, a 6-10 membered aryl ring, a 5-10 membered heteroaryl ring having 1-5 heteroatoms, and a 3-10 membered heterocyclic ring having 1-5 heteroatoms, or:

two or more R′ groups are taken together with their intervening atoms to form an optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring having, in addition to the intervening atoms, 0-5 heteroatoms, wherein each monocyclic ring is independently an optionally substituted, saturated, partially saturated or aromatic 3-20 membered ring having 0-5 heteroatoms.

In some embodiments, R^(a) is R″. In some embodiments, R^(a) is —OR″.

In some embodiments, R″ is R′. In some embodiments, R″ is —C(O)R′. In some embodiments, R″ is —CO₂R′. In some embodiments, R″ is —SO₂R′.

In some embodiments, R′ is hydrogen. In some embodiments, R′ is not hydrogen.

In some embodiments, R^(a) is R′. In some embodiments, R^(a) is —OR′. In some embodiments, R^(a) is —H. In some embodiments, R^(a) is —OH.

In some embodiments, L¹ is a covalent bond. In some embodiments, L¹ is not a covalent bond.

In some embodiments, L¹ is optionally substituted C₁₋₆ alkylene. In some embodiments, L¹ is optionally substituted linear C₁₋₆ alkylene. In some embodiments, L¹ is optionally substituted —CH₂—. In some embodiments, L¹ is optionally substituted —CH₂CH₂—. In some embodiments, L¹ is optionally substituted —CH₂CH₂CH₂—. In some embodiments, L¹ is optionally substituted —CH₂CH₂CH₂CH₂—. In some embodiments, L¹ is optionally substituted —CH₂CH₂CH₂CH₂CH₂—. In some embodiments, L¹ is optionally substituted —CH₂CH₂CH₂CH₂CH₂CH₂—. In some embodiments, —CH₂— bonded to —C(O)H is unsubstituted. In some embodiments, —CH₂— bonded to —C(O)H is monosubstituted. In some embodiments, L¹ is substituted. In some embodiments, L¹ is unsubstituted. In some embodiments, L¹ is —CH₂—. In some embodiments, L¹ is —CH₂CH₂—. In some embodiments, L¹ is —CH₂CH₂CH₂—. In some embodiments, L¹ is —CH₂CH₂CH₂CH₂—. In some embodiments, L¹ is —CH₂CH₂CH₂CH₂CH₂—. In some embodiments, L¹ is —CH₂CH₂CH₂CH₂CH₂CH₂—.

In some embodiments, L² is a covalent bond. In some embodiments, L² is not a covalent bond.

In some embodiments, L² is optionally substituted C₁₋₆ alkylene. In some embodiments, L² is optionally substituted linear C₁₋₆ alkylene. In some embodiments, L² is optionally substituted —CH₂—. In some embodiments, L² is optionally substituted —CH₂CH₂—. In some embodiments, L² is optionally substituted —CH₂CH₂CH₂—. In some embodiments, L² is optionally substituted —CH₂CH₂CH₂CH₂—. In some embodiments, L² is optionally substituted —CH₂CH₂CH₂CH₂CH₂—. In some embodiments, L² is optionally substituted —CH₂CH₂CH₂CH₂CH₂CH₂—. In some embodiments, —CH₂— bonded to —C(O)H is unsubstituted. In some embodiments, —CH₂— bonded to —C(O)H is monosubstituted. In some embodiments, L² is substituted. In some embodiments, L² is unsubstituted. In some embodiments, L² is —CH₂—. In some embodiments, L² is —CH₂CH₂—. In some embodiments, L² is —CH₂CH₂CH₂—. In some embodiments, L² is —CH₂CH₂CH₂CH₂—. In some embodiments, L² is —CH₂CH₂CH₂CH₂CH₂—. In some embodiments, L² is —CH₂CH₂CH₂CH₂CH₂CH₂—.

In some embodiments, at least one of L¹ and L² is not a covalent bond.

In some embodiments, an aldehyde is CH₃CHO. In some embodiments, an aldehyde is CH₃CH₂CHO. In some embodiments, an aldehyde is CH₃CH₂CH₂CHO. In some embodiments, an aldehyde is CH₂OHCHO. In some embodiments, an aldehyde is CH₂OHCH₂CHO. In some embodiments, an aldehyde is CH₂OHCH₂CH₂CHO.

In some embodiments, an aldol product is CH₃CH(OH)CH₂C(O)COOH. In some embodiments, an aldol product is CH₃CH₂CH(OH)CH₂C(O)COOH. In some embodiments, an aldol product is CH₃CH₂CH₂CH(OH)CH₂C(O)COOH. In some embodiments, an aldol product is CH₂OHCH(OH)CH₂C(O)COOH. In some embodiments, an aldol product is CH₂OHCH₂CH(OH)CH₂C(O)COOH. In some embodiments, an aldol product is CH₂OHCH₂CH₂CH(OH)CH₂C(O)COOH.

In some embodiments, an aldol-dehydration product is CH₃CH═CHC(O)COOH. In some embodiments, an aldol-dehydration product is CH₃CH₂CH═CHC(O)COOH. In some embodiments, an aldol-dehydration product is CH₃CH₂CH₂CH═CHC(O)COOH. In some embodiments, an aldol-dehydration product is CH₂OHCH═CHC(O)COOH. In some embodiments, an aldol-dehydration product is CH₂OHCH₂CH═CHC(O)COOH. In some embodiments, an aldol-dehydration product is CH₂OH CH₂CH₂CH═CHC(O)COOH.

In some embodiments, an alkene reduction product is CH₃CH₂CH₂C(O)COOH. In some embodiments, an alkene reduction product is CH₃CH₂CH₂CH₂C(O)COOH. In some embodiments, an alkene reduction product is CH₃CH₂CH₂CH₂CH₂C(O)COOH. In some embodiments, an alkene reduction product is CH₂OHCH₂CH₂C(O)COOH. In some embodiments, an alkene reduction product is CH₂OHCH₂CH₂CH₂C(O)COOH. In some embodiments, an alkene reduction product is CH₂OHCH₂CH₂CH₂CH₂C(O)COOH.

In some embodiments, an alkene reduction product is converted into a carbonyl reduction product, either catalyzed by an enzyme, through biosynthesis, or through traditional organic synthesis without enzymatic catalysis. In some embodiments, an alkene reduction product comprises a carbonyl group, and the carbonyl group is converted to —CH(OH)—. In some embodiments, a method comprises contacting an alkene reduction product with a carbonyl reduction product biosynthesis polypeptide so that a carbonyl reduction product is produced, wherein:

the alkene reduction product comprises a carbonyl group; and

a carbonyl group of the alkene reduction product is converted to —CH(OH)—.

In some embodiments, a carbonyl reduction product biosynthesis polypeptide is or comprises a reductase. In some embodiments, a carbonyl reduction product biosynthesis polypeptide is or comprises a keto reductase as described herein. In some embodiments, a carbonyl reduction product biosynthesis polypeptide is or comprises a 2-keto acid-2-reductase as described herein. In some embodiments, such an enzyme is a 6-hydroxy-2-oxohexanoate-2-reductase as described herein. In some embodiments, such an enzyme is described in US20170044551, the enzymes of which are incorporated herein by reference.

In some embodiments, conversion of an alkene reduction product into a carbonyl reduction product is catalyzed by a carbonyl reduction product biosynthesis polypeptide.

As for many other biosynthesis polypeptides, carbonyl reduction product biosynthesis polypeptides may be in organisms such as bacteria, may be engineered, and/or may be expressed at increased at increased protein and/or activity levels, and their products may be generated at increased rates and/or yields and/or substrates utilization.

In some embodiments, a carbonyl reduction product has the structure of formula P-4:

R^(a)-L²-L¹-CH₂—CH₂—CH(OH)—C(O)OH,   P-4

or a salt thereof, wherein each variable is independently as described herein.

In some embodiments, a carbonyl reduction product is CH₃CH₂CH₂CH(OH)COOH. In some embodiments, a carbonyl reduction product is CH₃CH₂CH₂CH₂CH(OH)COOH. In some embodiments, a carbonyl reduction product is CH₃CH₂CH₂CH₂CH₂CH(OH)COOH. In some embodiments, a carbonyl reduction product is CH₂OHCH₂CH₂CH(OH)COOH. In some embodiments, a carbonyl reduction product is CH₂OHCH₂CH₂CH₂CH(OH)COOH. In some embodiments, a carbonyl reduction product is CH₂OHCH₂CH₂CH₂CH₂CH(OH)COOH.

In some embodiments, a carbonyl reduction product is converted into a CoA transfer product, either catalyzed by an enzyme, through biosynthesis, or through traditional organic synthesis without enzymatic catalysis. In some embodiments, a CoA transfer product is a compound of formula P-5:

R^(a)-L²-L¹-CH₂—CH₂—CH(OH)—C(O)—S—CoA,   P-5

or a salt thereof, wherein each variable is independently as described herein.

In some embodiments, such a conversion is catalyzed by a CoA (CoA=Coenzyme A) transfer product biosynthesis polypeptide. In some embodiments, a CoA transfer product biosynthesis polypeptide is or comprises a CoA transferase as described herein, e.g., 2,6-dihydroxy-hexanoate CoA-transferase. In some embodiments, a CoA transferase is one described in US20170044551, the CoA transferases of which are incorporated herein by reference. In some embodiments, such a conversion is catalyzed by a CoA transfer product biosynthesis polypeptide.

As for many other biosynthesis polypeptides, CoA transfer product biosynthesis polypeptides may be in organisms such as bacteria, may be engineered, and/or may be expressed at increased at increased protein and/or activity levels, and their products may be generated at increased rates and/or yields and/or substrates utilization.

In some embodiments, a CoA transfer product is CH₃CH₂CH₂CH(OH)C(O)S-CoA. In some embodiments, a CoA transfer product is CH₃CH₂CH₂CH₂CH(OH)C(O)S-COA. In some embodiments, a CoA transfer product is CH₃CH₂CH₂CH₂CH₂CH(OH)C(O)S-COA. In some embodiments, a CoA transfer product is CH₂OHCH₂CH₂CH(OH)C(O)S-COA. In some embodiments, a CoA transfer product is CH₂OHCH₂CH₂CH₂CH(OH)C(O)S-COA. In some embodiments, a CoA transfer product is CH₂OHCH₂CH₂CH₂CH₂CH(OH)C(O)S-COA.

In some embodiments, a CoA transfer product is converted into a dehydration product, either catalyzed by an enzyme, through biosynthesis, or through traditional organic synthesis without enzymatic catalysis. In some embodiments, a dehydration product is a compound of formula P-6:

R^(a)-L²-L¹-CH₂—CH═CH—C(O)—S—CoA,   P-6

or a salt thereof, wherein each variable is independently as described herein.

In some embodiments, such a conversion is catalyzed by a dehydration product biosynthesis polypeptide. In some embodiments, a dehydration product biosynthesis polypeptide is or comprises a dehydratase as described herein. In some embodiments, a dehydratase is or comprises a 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase as described herein. In some embodiments, a dehydratase is described in US20170044551, the dehydratases of which is incorporated by reference.

In some embodiments, such a conversion is catalyzed by a dehydration product biosynthesis polypeptide.

As for many other biosynthesis polypeptides, dehydration product biosynthesis polypeptides may be in organisms such as bacteria, may be engineered, and/or may be expressed at increased at increased protein and/or activity levels, and their products may be generated at increased rates and/or yields and/or substrates utilization.

In some embodiments, a dehydration product is CH₃CH₂CH═CHC(O)S-CoA. In some embodiments, a dehydration product is CH₃CH₂CH₂CH═CHC(O)S-COA. In some embodiments, a dehydration product is CH₃CH₂CH₂CH₂CH═CHC(O)S-COA. In some embodiments, a dehydration product is CH₂OHCH₂CH═CHC(O)S-COA. In some embodiments, a dehydration product is CH₂OHCH₂CH₂CH═CHC(O)S-COA. In some embodiments, a dehydration product is CH₂OHCH₂CH₂CH₂CH═CHC(O)S-COA.

In some embodiments, a dehydration product, e.g. a compound of formula P-6 or a salt thereof, is converted into a reduction product, either catalyzed by an enzyme, through biosynthesis, or through traditional organic synthesis without enzymatic catalysis. In some embodiments, a reduction product is a compound of formula P-7:

R^(a)-L²-L¹-CH₂—CH₂—CH₂—C(O)—S—CoA,   P-7

or a salt thereof, wherein each variable is independently as described herein.

In some embodiments, such a conversion is catalyzed by a reduction product biosynthesis polypeptide. In some embodiments, a reduction product biosynthesis polypeptide is or comprises a 2,3-enoyl-CoA reductase, 2,3-dehydro-carboxyl CoA 2′3-reductase, e.g., 2,3-dehydro-hexanoyl-CoA 2,3-reductase as described herein. In some embodiments, a suitable reductase is described in US20170044551, the reductases of which are incorporated herein by reference. In some embodiments, such a conversion is catalyzed by a reduction product biosynthesis polypeptide.

As for many other biosynthesis polypeptides, reduction product biosynthesis polypeptides may be in organisms such as bacteria, may be engineered, and/or may be expressed at increased at increased protein and/or activity levels, and their products may be generated at increased rates and/or yields and/or substrates utilization.

In some embodiments, a reduction product is CH₃CH₂CH₂CH₂C(O)S-CoA. In some embodiments, a reduction product is CH₃CH₂CH₂CH₂CH₂C(O)S-COA. In some embodiments, a reduction product is CH₃CH₂CH₂CH₂CH₂CH₂C(O)S-COA. In some embodiments, a reduction product is CH₂OHCH₂CH₂CH₂C(O)S-COA. In some embodiments, a reduction product is CH₂OHCH₂CH₂CH₂CH₂C(O)S-COA. In some embodiments, a reduction product is CH₂OHCH₂CH₂CH₂CH₂CH₂C(O)S-COA.

In some embodiments, a reduction product, e.g. a compound of formula P-7 or a salt thereof, is converted into a CoA transfer product, either catalyzed by an enzyme, through biosynthesis, or through traditional organic synthesis without enzymatic catalysis. In some embodiments, a CoA transfer product is a compound of formula P-8:

R^(a)-L²-L¹-CH₂—CH₂—CH₂—C(O)—OH,   P-8

or a salt thereof, wherein each variable is independently as described herein.

In some embodiments, such a conversion is catalyzed by a CoA transfer product biosynthesis polypeptide. In some embodiments, a CoA transfer product biosynthesis polypeptide is or comprises a CoA transferase as described herein, e.g., a 6-hydroxyhexanoyl-CoA transferase as described herein. In some embodiments, a CoA transferase is described in US20170044551, the CoA transferases of which are incorporated herein by reference. In some embodiments, such a conversion is catalyzed by a CoA transfer product biosynthesis polypeptide.

As for many other biosynthesis polypeptides, CoA transfer product biosynthesis polypeptides may be in organisms such as bacteria, may be engineered, and/or may be expressed at increased at increased protein and/or activity levels, and their products may be generated at increased rates and/or yields and/or substrates utilization.

In some embodiments, a CoA transfer product is CH₃CH₂CH₂CH₂C(O)OH. In some embodiments, a CoA transfer product is CH₃CH₂CH₂CH₂CH₂C(O)OH. In some embodiments, a CoA transfer product is CH₃CH₂CH₂CH₂CH₂CH₂C(O)OH. In some embodiments, a CoA transfer product is CH₂OHCH₂CH₂CH₂C(O)OH. In some embodiments, a CoA transfer product is CH₂OHCH₂CH₂CH₂CH₂C(O)OH. In some embodiments, a CoA transfer product is CH₂OHCH₂CH₂CH₂CH₂CH₂C(O)OH.

In some embodiments, a CoA transfer product, e.g. a compound of formula P-8 or a salt thereof wherein R^(a) is —OH, is converted into an oxidation product, either catalyzed by an enzyme, through biosynthesis, or through traditional organic synthesis without enzymatic catalysis. In some embodiments, an oxidation product is a compound of formula P-9:

H—C(O)-L^(2′)-L¹-CH₂—CH₂—CH₂—C(O)—OH,   P-9

or a salt thereof, wherein L^(2′) is a covalent bond, or a bivalent, optionally substituted, linear or branched C₁₋₁₉ aliphatic or C₁₋₁₉ heteroaliphatic, wherein one or more methylene units are optionally and independently replaced by —C≡C—, —C(R″)₂—, -Cy-, —O—, —S—, —S—S—, —N(R″)—, —C(O)—, —C(S)—, —C(NR″)—, —C(O)N(R″)—, —N(R″)C(O)N(R″)—, —N(R″)C(O)O—, —S(O)—, —S(O)₂—, —S(O)₂N(R″)—, —C(O)S—, or —C(O)O—, and each other variable is independently as described herein.

In some embodiments, L^(2′) is a covalent bond. In some embodiments, L^(2′) is not a covalent bond. In some embodiments, at least one of L¹ and L^(2′) is not a covalent bond.

In some embodiments, L^(2′) is optionally substituted C₁₋₆ alkylene. In some embodiments, L^(2′) is optionally substituted linear C₁₋₆ alkylene. In some embodiments, L^(2′) is optionally substituted —CH₂—. In some embodiments, L^(2′) is optionally substituted —CH₂CH₂—. In some embodiments, L^(2′) is optionally substituted —CH₂CH₂CH₂—. In some embodiments, L^(2′) is optionally substituted —CH₂CH₂CH₂CH₂—. In some embodiments, L^(2′) is optionally substituted —CH₂CH₂CH₂CH₂CH₂—. In some embodiments, L^(2′) is optionally substituted —CH₂CH₂CH₂CH₂CH₂CH₂—. In some embodiments, —CH₂— bonded to —C(O)H is unsubstituted. In some embodiments, —CH₂— bonded to —C(O)H is monosubstituted. In some embodiments, L^(2′) is substituted. In some embodiments, L^(2′) is unsubstituted. In some embodiments, L^(2′) is —CH₂—. In some embodiments, L^(2′) is —CH₂CH₂—. In some embodiments, L^(2′) is —CH₂CH₂CH₂—. In some embodiments, L^(2′) is —CH₂CH₂CH₂CH₂—. In some embodiments, L^(2′) is —CH₂CH₂CH₂CH₂CH₂—. In some embodiments, L^(2′) is —CH₂CH₂CH₂CH₂CH₂CH₂—.

In some embodiments, such a conversion is catalyzed by an oxidation product biosynthesis polypeptide. In some embodiments, an oxidation product biosynthesis polypeptide is or comprises an alcohol dehydrogenase, e.g., a primary alcohol dehydrogenase such as 6-hydroxyhexanoate dehydrogenase, as described herein. In some embodiments, an alcohol dehydrogenase is described in US20170044551, the alcohol dehydrogenases of which are incorporated herein by reference. In some embodiments, such a conversion is catalyzed by an oxidation product biosynthesis polypeptide.

As for many other biosynthesis polypeptides, oxidation product biosynthesis polypeptides may be in organisms such as bacteria, may be engineered, and/or may be expressed at increased at increased protein and/or activity levels, and their products may be generated at increased rates and/or yields and/or substrates utilization.

In some embodiments, an oxidation product is HC(O)CH₂CH₂CH₂C(O)OH. In some embodiments, an oxidation product is HC(O)CH₂CH₂CH₂CH₂C(O)OH. In some embodiments, an oxidation product is HC(O)CH₂CH₂CH₂CH₂CH₂C(O)OH.

In some embodiments, an oxidation product, e.g. a compound of formula P-9 or a salt thereof, is converted into an aldehyde oxidation product, either catalyzed by an enzyme, through biosynthesis, or through traditional organic synthesis without enzymatic catalysis. In some embodiments, an oxidation product is a compound of formula P-10:

HO—C(O)-L^(2′)-L¹-CH₂—CH₂—CH₂—C(O)—OH,   P-10

or a salt thereof, wherein each other variable is independently as described herein.

In some embodiments, such a conversion is catalyzed by an aldehyde oxidation product biosynthesis polypeptide. In some embodiments, an aldehyde oxidation product biosynthesis polypeptide is or comprises an aldehyde dehydrogenase, e.g., a 6-hydroxyhexanoate dehydrogenase, as described herein. In some embodiments, an aldehyde dehydrogenase is described in US20170044551, the aldehyde dehydrogenases of which are incorporated herein by reference. In some embodiments, such a conversion is catalyzed by an aldehyde oxidation product biosynthesis polypeptide.

As for many other biosynthesis polypeptides, aldehyde oxidation product biosynthesis polypeptides may be in organisms such as bacteria, may be engineered, and/or may be expressed at increased at increased protein and/or activity levels, and their products may be generated at increased rates and/or yields and/or substrates utilization.

In some embodiments, an aldehyde oxidation product is HOC(O)CH₂CH₂CH₂C(O)OH. In some embodiments, an oxidation product is HOC(O)CH₂CH₂CH₂CH₂C(O)OH. In some embodiments, an oxidation product is HOC(O)CH₂CH₂CH₂CH₂CH₂C(O)OH.

In some embodiments, a CoA transfer product, e.g. a compound of formula P-8 or a salt thereof, is converted into a carboxyl reduction product, either catalyzed by an enzyme, through biosynthesis, or through traditional organic synthesis without enzymatic catalysis. In some embodiments, a carboxyl reduction product is a compound of formula P-9′:

R^(a)-L²-L¹-CH₂—CH₂—CH₂—C(O)—H,   P-9′

or a salt thereof, wherein each variable is independently as described herein.

In some embodiments, such a conversion is catalyzed by a carboxyl reduction product biosynthesis polypeptide. In some embodiments, a carboxyl reduction product biosynthesis polypeptide is or comprises a carboxylic acid reductase or aldehyde dehydrogenase as described herein. In some embodiments, a carboxyl reduction product biosynthesis polypeptide is or comprises a 6-hydroxyhexanoate 1-reductase. In some embodiments, a carboxyl reduction product biosynthesis polypeptide is or comprises a carboxylic acid reductase or aldehyde dehydrogenase described in US20170044551, the carboxylic acid reductases or aldehyde dehydrogenases of which are incorporated herein by reference. In some embodiments, such a conversion is catalyzed by a carboxyl reduction product biosynthesis polypeptide.

As for many other biosynthesis polypeptides, carboxyl reduction product biosynthesis polypeptides may be in organisms such as bacteria, may be engineered, and/or may be expressed at increased at increased protein and/or activity levels, and their products may be generated at increased rates and/or yields and/or substrates utilization.

In some embodiments, a carboxyl reduction product is CH₃CH₂CH₂CH₂C(O)H. In some embodiments, a carboxyl reduction product is CH₃CH₂CH₂CH₂CH₂C(O)H. In some embodiments, a carboxyl reduction product is CH₃CH₂CH₂CH₂CH₂CH₂C(O)H. In some embodiments, a carboxyl reduction product is CH₂OHCH₂CH₂CH₂C(O)H. In some embodiments, a carboxyl reduction product is CH₂OHCH₂CH₂CH₂CH₂C(O)H. In some embodiments, a carboxyl reduction product is CH₂OHCH₂CH₂CH₂CH₂CH₂C(O)H.

In some embodiments, a carboxyl reduction product, e.g. a compound of formula P-9′ or a salt thereof, is converted into an aldehyde reduction product, either catalyzed by an enzyme, through biosynthesis, or through traditional organic synthesis without enzymatic catalysis. In some embodiments, an aldehyde reduction product is a compound of formula P-10′:

R^(a)-L²-L¹-CH₂—CH₂—CH₂—CH₂—OH,   P-10′

or a salt thereof, wherein each variable is independently as described herein.

In some embodiments, such a conversion is catalyzed by an aldehyde reduction product biosynthesis polypeptide. In some embodiments, an aldehyde reduction product biosynthesis polypeptide is or comprises an aldehyde reductase or an alcohol (e.g., primary alcohol) dehydrogenase as described herein. In some embodiments, an aldehyde reductase or an alcohol (e.g., primary alcohol) dehydrogenase is described in US20170044551, the reductases and dehydrogenases of which are incorporated herein by reference. In some embodiments, such a conversion is catalyzed by an aldehyde reduction product biosynthesis polypeptide.

As for many other biosynthesis polypeptides, aldehyde reduction product biosynthesis polypeptides may be in organisms such as bacteria, may be engineered, and/or may be expressed at increased at increased protein and/or activity levels, and their products may be generated at increased rates and/or yields and/or substrates utilization.

In some embodiments, an aldehyde reduction product is CH₃CH₂CH₂CH₂CH₂OH. In some embodiments, an aldehyde reduction product is CH₃CH₂CH₂CH₂CH₂CH₂OH. In some embodiments, an aldehyde reduction product is CH₃CH₂CH₂CH₂CH₂CH₂CH₂OH. In some embodiments, an aldehyde reduction product is CH₂OHCH₂CH₂CH₂CH₂OH. In some embodiments, an aldehyde reduction product is CH₂OHCH₂CH₂CH₂CH₂CH₂OH. In some embodiments, an aldehyde reduction product is CH₂OHCH₂CH₂CH₂CH₂CH₂CH₂OH.

In some embodiments, an alkene reduction product, e.g. a compound of formula P-3 or a salt thereof, is converted into a decarboxylation product, either catalyzed by an enzyme, through biosynthesis, or through traditional organic synthesis without enzymatic catalysis. In some embodiments, a decarboxylation product is a compound of formula P-4′:

R^(a)-L²-L¹-CH₂—CH₂—C(O)—H,   P-4′

or a salt thereof, wherein each variable is independently as described herein.

In some embodiments, such a conversion is catalyzed by a decarboxylation product biosynthesis polypeptide. In some embodiments, a decarboxylation product biosynthesis polypeptide is or comprises a decarboxylase as described herein. In some embodiments, a decarboxylase is a 2-keto-acid decarboxylase as described herein. In some embodiments, a decarboxylase is described in US20170044551, the decarboxylases of which are incorporated herein by reference. In some embodiments, such a conversion is catalyzed by a decarboxylation product biosynthesis polypeptide.

As for many other biosynthesis polypeptides, decarboxylation product biosynthesis polypeptides may be in organisms such as bacteria, may be engineered, and/or may be expressed at increased at increased protein and/or activity levels, and their products may be generated at increased rates and/or yields and/or substrates utilization.

In some embodiments, a decarboxylation product is CH₃CH₂CH₂CHO. In some embodiments, a decarboxylation product is CH₃CH₂CH₂CH₂CHO. In some embodiments, a decarboxylation product is CH₃CH₂CH₂CH₂CH₂CHO. In some embodiments, a decarboxylation product is CH₂OHCH₂CH₂CHO. In some embodiments, a decarboxylation product is CH₂OHCH₂CH₂CH₂CHO. In some embodiments, a decarboxylation product is CH₂OHCH₂CH₂CH₂CH₂CHO.

In some embodiments, a decarboxylation product, e.g. a compound of formula P-4′ or a salt thereof, is converted into an aldehyde reduction product, either catalyzed by an enzyme, through biosynthesis, or through traditional organic synthesis without enzymatic catalysis. In some embodiments, an aldehyde reduction product is a compound of formula P-5′:

R^(a)-L²-L¹-CH₂—CH₂—CH₂—OH,   P-5′

or a salt thereof, wherein each variable is independently as described herein.

In some embodiments, such a conversion is catalyzed by an aldehyde reduction product biosynthesis polypeptide. In some embodiments, an aldehyde reduction product biosynthesis polypeptide is or comprises a primary alcohol dehydrogenase as described herein. In some embodiments, a primary alcohol dehydrogenase is described in US20170044551, the primary alcohol dehydrogenase of which are incorporated herein by reference. In some embodiments, such a conversion is catalyzed by an aldehyde reduction product biosynthesis polypeptide.

As for many other biosynthesis polypeptides, aldehyde reduction product biosynthesis polypeptides may be in organisms such as bacteria, may be engineered, and/or may be expressed at increased at increased protein and/or activity levels, and their products may be generated at increased rates and/or yields and/or substrates utilization.

In some embodiments, an aldehyde reduction product is CH₃CH₂CH₂CH₂OH. In some embodiments, an aldehyde reduction product is CH₃CH₂CH₂CH₂CH₂OH. In some embodiments, an aldehyde reduction product is CH₃CH₂CH₂CH₂CH₂CH₂OH. In some embodiments, an aldehyde reduction product is CH₂OHCH₂CH₂CH₂OH. In some embodiments, an aldehyde reduction product is CH₂OHCH₂CH₂CH₂CH₂OH. In some embodiments, an aldehyde reduction product is CH₂OHCH₂CH₂CH₂CH₂CH₂OH.

In some embodiments, the present disclosure provides nucleic acids encoding one or more biosynthesis polypeptides. In some embodiments, such nucleic acids comprise unnatural sequences. In some embodiments, such nucleic acids are optimized for expression in production organisms, e.g., bacteria.

As demonstrated herein, various technologies are available for assess activities of polypeptides for biosynthesis activities. For example, various technologies for assessing activities of aldol-dehydration product biosynthesis polypeptides (e.g., hydratase-aldolases) or alkene reduction product biosynthesis polypeptides (e.g., enzymes for reducing aldol-dehydration products) are described in the Examples.

In some embodiments, various biosynthesis polypeptides, e.g., an aldol-dehydration product biosynthesis polypeptide, are in organisms, in many embodiments, microorganisms such as bacteria, fungi, etc. In some embodiments, they are expressed from one or more recombinant nucleic acids. In some embodiments, various transformations are performed biosynthetically, e.g., in organisms such as bacteria. In some embodiments, organisms (e.g., microbes such as bacteria) are engineered to contain exogenous nucleic acids that encode biosynthetic polypeptides, e.g., aldol-dehydration product biosynthesis polypeptides such as hydratase-aldolases.

In some embodiments, organism, e.g., those engineered for producing aldol-dehydration products, express modulated levels, typically increased levels and/or activities of aldol-dehydration product biosynthesis polypeptides such as hydratase-aldolase polypeptides.

In some embodiments, organisms comprise engineered nucleic acids and/or express engineered biosynthesis polypeptides, e.g., aldol-dehydration product biosynthesis polypeptides (e.g., various hydratase-aldolases). In some embodiments, an engineered nucleic acid comprises one or more sequence difference compared to a reference nucleic acid. In some embodiments, a reference nucleic acid is a corresponding nucleic acid in an organism to which an engineered nucleic acid is introduced. In some embodiments, a reference nucleic acid is a natural nucleic acid. In some embodiments, an engineered nucleic acid encodes the same polypeptide or a characteristic element thereof as a reference nucleic acid, e.g., a natural nucleic acid. In some embodiments, an engineered nucleic acid encodes a polypeptide or a characteristic element thereof which is different than that encoded by as a reference nucleic acid. In some embodiments, an engineered polypeptide comprises one or more differences compared to a reference polypeptide (e.g., encoded by a reference nucleic acid, found in nature, etc.). In some embodiments, an engineered polypeptide comprises one or more different amino acid residues compared to a reference polypeptide. In some embodiments, an engineered polypeptide is a polypeptide which is absent from an organism to which it is introduced. In some embodiments, an engineered polypeptide is homologous to a reference polypeptide, e.g., sharing 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 95%, 99% or more homology with a reference polypeptide or a characteristic element thereof. In some embodiments, a characteristic element is a domain which catalyzes a relevant reaction. In some embodiments, a characteristic element is a set of amino acid residues. In some embodiments, a characteristic element is a set of amino acid residues that form contact with substrates, products, co-factors, etc. and/or promotes a relevant reaction. As appreciated by those skilled in the art, residues in a set of amino acid residues can be next to each other in sequence, or can be separated. In some embodiments, two or more amino acid residues in a set may be spatially close to each other, e.g., in a catalytic pocket.

In some embodiments, for biosynthetic productions, organisms may express high levels and/or activities of one or more biosynthetic polypeptides. In some embodiments, an organism provides an increased rate and/or yield for producing a desired product.

As described herein, in some embodiments, the present disclosure provides high product yields. In some embodiments, a yield, e.g., of a one or multiple step process involving one or more biosynthesis polypeptides, is about or at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 mg/L, or is about or at least about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.7, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 250, or 300 g/L. In some embodiments, provided technologies provide high utilization of a substrate, e.g., pyruvate, for a desired product. In some embodiments, the utilization percentage for a desired product is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.

Those skilled in the art appreciate that various compounds of the present disclosure, e.g., compounds of formula P-1, P-2, P-3, P-4, P-4′, P-5, P-5′, P-6, P-7, P-8, P-9, P-9′, P-10, or P-10′, or salts thereof, are useful as materials for production of various compounds, materials and products. For example, adipic acid can be used to produce nylon 6,6, polyester polyols, polyester resins, plasticizers, foods, and other materials. 1,5-Pentanediol can be used to manufacture various polyurethanes, polyester polyols, and polyesters. 1,6-Hexanediol (HDO) can be used to manufacture various polyesters, some of which are useful for industrial coating applications. HDO can also be utilized to produce polyurethane, which among other things can be used as coatings for automotive applications. In some embodiments, HDO is used for production of macrodiols, for example, adipate esters and polycarbonate diols used in, e.g., elastomers and polyurethane dispersions (e.g., for parquet flooring and leather coatings). Through traditional chemical or through biosynthesis processes or combinations thereof, 6-hydroxy hexanoic acid can be cyclized to make ε-caprolactone which can then be aminated to make ε-caprolactam. Through traditional chemical or through biosynthesis processes or combinations thereof, 6-hydroxy hexanoic acid can be aminated to make 6-amino hexanoic acid which can then be cyclized to make ε-caprolactam. ε-Caprolactam, among other things, can be used for the production of Nylon6, a widely used polymer in many different industries. ε-Caprolactone can be polymerized to make polycaprolactone (PCL) a biodegradable polyester with various applications including for the production of specialty polyurethanes. Various 2-ketocarboxylic acids are useful for various industrial relevant chemicals and pharmaceuticals. In some embodiments, such chemicals and pharmaceuticals, or intermediates thereof, are amino acids or α-hydroxy carboxylic acids. In some embodiments, compounds of the present disclosure are utilized to manufacture polyesters, polyester polyols, polyurethane, nylon (e.g., from adipic acid), polycarbonate diols (e.g., from HDO or 1,5-pentanediol, etc.), diacrylate esters (e.g., from HDO or 1,5-pentanediol, etc.), diglycidyl ethers (e.g., from HDO or 1,5-pentanediol, etc.), etc.

In some embodiments, the present disclosure provides preparations of provided processes, e.g., preparations of compounds of formula P-1, P-2, P-3, P-4, P-4′, P-5, P-5′, P-6, P-7, P-8, P-9, P-9′, P-10, or P-10′, or salts thereof, and various compounds, materials, products, etc., prepared from such compounds.

Provided technologies provide a number of advantages. Among other things, provided processes utilize one or more biosynthesis polypeptides and/or materials from renewable sources, which can improve efficiency and/or reduce pollution. In some embodiments, preparations of the present disclosure (e.g., of compounds of formula P-1, P-2, P-3, P-4, P-4′, P-5, P-5′, P-6, P-7, P-8, P-9, P-9′, P-10, or P-10′, or salts thereof, and various compounds, materials, products, etc., prepared from such compounds) comprise enriched levels of one or more isotopes, e.g., ¹⁴C, compared to those prepared from fossil carbon sources. In some embodiments, preparations using fossil carbon sources have a ¹⁴C level of 0 or virtually 0. Technologies for assessing isotopic ratios and/or levels of various atoms in compounds, compositions, preparations products, etc., are well known to those skilled in the art and can be utilized in accordance with the present disclosure. For example, in some embodiments, isotopic enrichment can be readily assessed by mass spectrometry using techniques such as accelerated mass spectrometry (AMS) and/or Stable Isotope Ratio Mass Spectrometry (SIRMS), and/or by Site-Specific Natural Isotopic Fractionation by Nuclear Magnetic Resonance (SNIF-NMR).

As appreciated by those skilled in the art, provided methods can be performed in vitro in a system comprising one or more biosynthesis polypeptides. In many embodiments, provided technologies are performed using organisms, e.g., microorganisms such as bacteria, that express one or more biosynthesis polypeptides. In some embodiments, the present disclosure provides organisms, e.g., bacteria, that express one or more biosynthesis polypeptides as described herein. In some embodiments, such organisms are engineered. In some embodiments, such organisms are engineered and/or cultured to express increased levels of proteins and/or activities of one or more biosynthesis polypeptides. In some embodiments, such organisms are engineered and/or cultured to utilize carbon sources to more efficiently produce desired products.

In some embodiments, the present disclosure provides an organism that produces an aldol product of an aliphatic aldehyde, the microbe comprising increased expression or activity of an aldol product biosynthesis polypeptide. In some embodiments, an organism is engineered. In some embodiments, an organism is a bacterium.

In some embodiments, the present disclosure provides an organism that produces an aldol-dehydration product of an aldehyde, the microbe comprising increased expression or activity of an aldol product biosynthesis polypeptide, an aldol-dehydration product biosynthesis polypeptide, a dehydration product biosynthesis polypeptide, and combinations thereof. In some embodiments, the present disclosure provides an organism that produces an aldol-dehydration product of an aldehyde, the microbe comprises increased expression or activity of an aldol-dehydration product biosynthesis polypeptide. In some embodiments, an organism is engineered. In some embodiments, an organism is a bacterium. In some embodiments, an aldehyde is an aliphatic aldehyde.

In some embodiments, the present disclosure provides an organism that produces an alkene reduction product, the microbe comprising increased expression or activity of an alkene reduction product biosynthesis polypeptide. In some embodiments, the present disclosure provides an organism that produces an alkene reduction product from pyruvate and an aldehyde, the microbe comprising increased expression or activity of an alkene reduction product biosynthesis polypeptide. In some embodiments, an organism is engineered. In some embodiments, an organism is a bacterium.

In some embodiments, the present disclosure provides cultures of organisms as described herein. In some embodiments, the present disclosure provides cultures of bacteria. In some embodiments, a culture comprises one or more products of one or more biosynthesis polypeptides, e.g., one or more compounds of formula P-1, P-2, P-3, P-4, P-4′, P-5, P-5′, P-6, P-7, P-8, P-9, P-9′, P-10, or P-10′, or salts thereof.

As appreciated by those skilled in the art, pyruvate may be provided as pyruvic acid or a salt thereof.

In one aspect, provided herein is a method for preparing a compound of Formula I:

wherein R is CH₂OH, CH₃ or H, or a salt thereof, or a solvate of the compound or the salt, wherein the method comprises enzymatic steps.

In some embodiments, the method comprises, or alternatively consists essentially of, or yet further consists of, combining or incubating a C_(N) aldehyde of formula

wherein R is CH₂OH, CH₃ or H, and a pyruvate in a solution under conditions that (a) convert the C_(N) aldehyde and the pyruvate to a C_(N+3) 3,4-dehydro-2-keto-carboxylic acid intermediate through an aldol condensation reaction catalyzed by a hydratase-aldolase having an EC number 4.1.2.45 or EC number 4.1.2.34 or EC number 4.1.1.4 (referred herein as Ads-Hyd); and then (b) convert the C_(N+3) 3,4-dehydro-2-keto-carboxylic acid to C_(N+3) 2-keto-carboxylic acid (i.e., the compound of Formula I), or salt thereof, or a solvate of the compound or the salt, using a oxidoreductase having an EC number 1.6.5. (e.g., EC number 1.6.5.5.).

In some embodiments, the method comprises, or alternatively consists essentially of, or yet further consists of, combining or incubating a C_(N) aldehyde of formula

wherein R is CH₂OH, CH₃ or H, and a pyruvate in a solution under conditions that (a) convert the C_(N) aldehyde and the pyruvate first to a C_(N+3) 4-hydroxy-2-keto-carboxylic acid intermediate through an aldol addition reaction catalyzed by a hydratase-aldolase having an EC number 4.1.2.45 or EC number 4.1.2.34 or EC number 4.1.1.4 (referred herein as Ads-Hyd); then (b) convert 4-hydroxy-2-keto-carboxylic acid to C_(N+3) 3,4-dehydro-2-keto-carboxylic acid using the hydratase-aldolase; and then (c) convert the C_(N+3) 3,4-dehydro-2-keto-carboxylic acid to C_(N+3) 2-keto-carboxylic acid (i.e., the compound of Formula I), or salt thereof, or a solvate of the compound or the salt, using a oxidoreductase having an EC number 1.6.5. (e.g., EC number 1.6.5.5.)

In another aspect, provided herein is a method for preparing a compound selected from 1,5-pentanediol, adipic acid, 1,6-hexanediol, and 6-hydroxy hexanoic acid, said method comprising, or alternatively consisting essentially of, or yet further consisting of: a) converting a 3-hydroxy-propanal and a pyruvate to a 6-hydroxy-2-keto carboxylic acid intermediate using a combination of a hydratase-aldolase having an EC number 4.1.2.45 or EC number 4.1.2.34 or EC number 4.1.1.4 and a oxidoreductase having an EC number 1.6.5 (e.g., EC number 1.6.5.5); and b) converting the 6-hydroxy-2-keto carboxylic acid intermediate to the compound through enzymatic steps.

In some embodiments, the hydratase-aldolase is a trans-o-hydroxybenzylidenepyruvate hydratase-aldolase having an EC number 4.1.2.45. In some embodiments, the hydratase-aldolase is a trans-2′-carboxybenzalpyruvate hydratase-aldolase having an EC number 4.1.2.34. In some embodiments, the hydratase-aldolase is a Acetoacetate decarboxylase having an EC number 4.1.1.4.

In some embodiments, a microorganism is used as a host for the preparation of a compound of Formula I, or a compound selected from 1,5-pentanediol, adipic acid, 1,6-hexanediol, and 6-hydroxy hexanoic acid, or a salt thereof, or a solvate of the compound or the salt. As used herein, a “host” refers to a cell or microorganism that can produce one or more enzymes capable of catalyzing a reaction either inside (by, e.g., uptaking the starting material(s) and optionally secreting the product(s)) or outside (by, e.g., secreting the enzyme) the cell or microorganism.

In some embodiments, the method further comprises or alternatively consists essentially of, or yet further consists of, isolating the compound selected from 1,5-pentanediol, adipic acid, 1,6-hexanediol, and 6-hydroxy hexanoic acid or a salt thereof, or a solvate of the compound or the salt from the solution, culture, and/or the host cell.

In some embodiments, the conditions of the methods disclosed herein comprise or alternatively consist essentially of, or yet further consist of, incubating or contacting the components at a temperature from about 10 to about 200° C., or alternatively at least (all temperatures provided in degrees Celsius) 10, 15, 20, 25, 28, 29, 30, 31, 32, 33, 34, 35, 37, 37, 38, 39, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180 or 190° C., or not higher than 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, or 25° C. with the lower temperature limit being 10° C. In some embodiments, the conditions or alternatively consists essentially of, or yet further consists of, the pH of the incubation solution is from about 2 to about 12. In some embodiments, the pH is at least 2, or 3, 4, 5, 5.5, 6, 6.5, 7, 7.5, 8, or 9 up to about 12. In some embodiments, the pH is not higher than 12, 11, 10, 9, 8, 7.5, 7, 6.5, 6, 5.5, or 4 with the lower pH limit being no lower than 2.

In some embodiments, the conditions comprise or alternatively consist essentially of, or yet further consist of, a molar concentration of pyruvate and C_(N) aldehyde are present at a concentration from about 0.1 μM to about 5 M. In some embodiments, the concentration is at least about 0.1, 0.5, 1, 10, 100, 500 μM or 1 M. In some embodiments, the concentration is not higher than about 4 M, 3 M, 2 M, 1 M, 500 μM, 200 μM, 100 μM, or 10 μM. The concentration of pyruvate and C_(N) can be independently the same or different and will vary with the other conditions of the incubation.

In some embodiments, the conditions comprise the presence of a non-natural microorganism that produces one or more enzymes selected from the group consisting of a class I/II pyruvate dependent aldolase, hydratase-aldolase, dehydratase, quinone oxidoreductase, enoyl-CoA reductase, primary alcohol dehydrogenase, keto-acid decarboxylase, coenzyme A transferase, and carboxylic acid reductase. Each of these enzymes is a reaction specific enzyme.

In some embodiments, the microorganism or host is genetically engineered to overexpress the enzymes or to express enzymes in an amount greater than the wild-type counterpart. Methods to determine the expression level of an enzyme or expression product are known in the art, e.g., by PCR.

In some embodiments, the C_(N) aldehyde is 3-hydroxy-propanal.

In some embodiments, the method further comprises or alternatively consists essentially of, or yet further consists of, preparing the 3-hydroxy-propanal and pyruvate from glycerol, C5 sugars, C6 sugars, phospho-glycerates, other carbon sources, intermediates of the glycolysis pathway, intermediates of propanoate metabolism, or combinations thereof.

In some embodiments, the 3-hydroxy-propanal is obtained through dehydration of glycerol.

In some embodiments, the C5 sugar comprises or alternatively consists essentially of, or yet further consists of, one or more of xylose, xylulose, ribulose, arabinose, lyxose, and ribose.

In some embodiments, the C6 sugar comprises or alternatively consists essentially of, or yet further consists of, one or more of allose, altrose, glucose, mannose, gulose, idose, talose, galactose, fructose, psicose, sorbose, and tagatose.

In some embodiments, the other carbon source is a feedstock suitable as a carbon source for a microorganism, wherein the feedstock comprises or alternatively consists essentially of, or yet further consists of, amino acids, lipids, corn stover, miscanthus, municipal waste, energy cane, sugar cane, bagasse, starch stream, dextrose stream, methanol, formate, or combinations thereof.

In some embodiments, a microorganism is used as a host for the preparation of 1,5-pentanediol, adipic acid, 1,6-hexanediol, or 6-hydroxy hexanoic acid.

In some embodiments, the microorganism has the ability to convert C5 sugars, C6 sugars, glycerol, other carbon sources, or a combination thereof to pyruvate.

In some embodiments, the microorganism is engineered for enhanced sugar uptake, e.g., C5 sugar uptake, simultaneous C6/C5 sugar uptake, simultaneous C6 sugar/glycerol uptake, simultaneous C5 sugar/glycerol uptake, or combinations thereof.

In another aspect, provided herein is a method for producing a 2-keto carboxylic acid of formula:

wherein R is H, CH₃, or CH₂OH; the method comprising, consisting essentially of, or consisting of contacting pyruvate and

with a hydratase-aldolase and a quinone oxidoreductase in a culture comprising one or more non-naturally occurring microbial organisms to produce the 2-keto carboxylic acid; wherein the hydratase-aldolase and the quinone oxidoreductase are expressed by the one or more non-naturally occurring microbial organisms.

In another aspect, provided herein is a method for producing a 2-keto carboxylic acid of formula:

wherein R is H, CH₃, or CH₂OH; the method comprising, consisting essentially of, or consisting of contacting pyruvate and

with a hydratase-aldolase and a quinone oxidoreductase in a culture comprising one or more non-naturally occurring microbial organisms to produce the 2-keto carboxylic acid; wherein the hydratase-aldolase and the quinone oxidoreductase are expressed by the one or more non-naturally occurring microbial organisms, and the method is performed in the presence of the one or more non-naturally occurring microbial organisms.

In another aspect, provided herein is a method for producing a 2-keto carboxylic acid of formula:

wherein R is H, CH₃, or CH₂OH; the method comprising, consisting essentially of, or consisting of contacting pyruvate and

with a trans-o-hydroxybenzylidenepyruvate hydratase-aldolase and a quinone oxidoreductase in a culture comprising one or more non-naturally occurring microbial organisms to produce the 2-keto carboxylic acid; wherein the trans-o-hydroxybenzylidenepyruvate hydratase-aldolase and the quinone oxidoreductase are expressed by the one or more non-naturally occurring microbial organisms, and the method is performed in the presence of the one or more non-naturally occurring microbial organisms.

In another aspect, provided herein is a method for producing a 2-keto carboxylic acid of formula:

wherein R is H, CH₃, or CH₂OH; the method comprising, consisting essentially of, or consisting of contacting pyruvate and

with a hydratase-aldolase and a quinone oxidoreductase in a culture comprising one or more non-naturally occurring microbial organisms to produce the 2-keto carboxylic acid; wherein the hydratase-aldolase and the quinone oxidoreductase are expressed by the one or more non-naturally occurring microbial organisms; and wherein the pyruvate and

undergo an aldol condensation reaction solely catalyzed by the hydratase-aldolase to produce a 2-oxo-3-enoic acid, and the 2-oxo-3-enoic acid undergoes a reduction solely catalyzed by the quinone oxidoreductase to produce the 2-keto carboxylic acid.

In another aspect, provided herein is a method for producing a 2-keto carboxylic acid of formula:

wherein R is H, CH₃, or CH₂OH; the method comprising, consisting essentially of, or consisting of contacting pyruvate and

with a hydratase-aldolase and a quinone oxidoreductase in a culture comprising one or more non-naturally occurring microbial organisms to produce the 2-keto carboxylic acid; wherein the hydratase-aldolase and the quinone oxidoreductase are expressed by the one or more non-naturally occurring microbial organisms, and the method is performed in the presence of the one or more non-naturally occurring microbial organisms; and wherein the pyruvate and

undergo an aldol condensation reaction solely catalyzed by the hydratase-aldolase to produce a 2-oxo-3-enoic acid, and the 2-oxo-3-enoic acid undergoes a reduction solely catalyzed by the quinone oxidoreductase to produce the 2-keto carboxylic acid.

In another aspect, provided herein is a method for producing a 2-keto carboxylic acid of formula:

wherein R is H, CH₃, or CH₂OH; the method comprising, consisting essentially of, or consisting of contacting pyruvate and

with a hydratase-aldolase and a quinone oxidoreductase in a culture comprising two or more non-naturally occurring microbial organisms to produce the 2-keto carboxylic acid; wherein the hydratase-aldolase and the quinone oxidoreductase are expressed by the two or more non-naturally occurring microbial organisms.

In another aspect, provided herein is a method for producing a 2-keto carboxylic acid of formula:

wherein R is H, CH₃, or CH₂OH; the method comprising, consisting essentially of, or consisting of contacting pyruvate and

with a hydratase-aldolase and a quinone oxidoreductase in a culture comprising two or more non-naturally occurring microbial organisms to produce the 2-keto carboxylic acid; wherein the hydratase-aldolase and the quinone oxidoreductase are expressed by the two or more non-naturally occurring microbial organisms, and the method is performed in the presence of the two or more non-naturally occurring microbial organisms.

In another aspect, provided herein is a method for producing a 2-keto carboxylic acid of formula:

wherein R is H, CH₃, or CH₂OH; the method comprising, consisting essentially of, or consisting of contacting pyruvate and

with a trans-o-hydroxybenzylidenepyruvate hydratase-aldolase and a quinone oxidoreductase in a culture comprising two or more non-naturally occurring microbial organisms to produce the 2-keto carboxylic acid; wherein the trans-o-hydroxybenzylidenepyruvate hydratase-aldolase and the quinone oxidoreductase are expressed by the two or more non-naturally occurring microbial organisms, and the method is performed in the presence of the two or more non-naturally occurring microbial organisms.

In another aspect, provided herein is a method for producing a 2-keto carboxylic acid of formula:

wherein R is H, CH₃, or CH₂OH; the method comprising, consisting essentially of, or consisting of contacting pyruvate and

with a hydratase-aldolase and a quinone oxidoreductase in a culture comprising two or more non-naturally occurring microbial organisms to produce the 2-keto carboxylic acid; wherein the hydratase-aldolase and the quinone oxidoreductase are expressed by the two or more non-naturally occurring microbial organisms; and wherein the pyruvate and

undergo an aldol condensation reaction solely catalyzed by the hydratase-aldolase to produce a 2-oxo-3-enoic acid, and the 2-oxo-3-enoic acid undergoes a reduction solely catalyzed by the quinone oxidoreductase to produce the 2-keto carboxylic acid.

In another aspect, provided herein is a method for producing a 2-keto carboxylic acid of formula:

wherein R is H, CH₃, or CH₂OH; the method comprising, consisting essentially of, or consisting of contacting pyruvate and

with a hydratase-aldolase and a quinone oxidoreductase in a culture comprising two or more non-naturally occurring microbial organisms to produce the 2-keto carboxylic acid; wherein the hydratase-aldolase and the quinone oxidoreductase are expressed by the two or more non-naturally occurring microbial organisms, and the method is performed in the presence of the two or more non-naturally occurring microbial organisms; and wherein the pyruvate and

undergo an aldol condensation reaction solely catalyzed by the hydratase-aldolase to produce a 2-oxo-3-enoic acid, and the 2-oxo-3-enoic acid undergoes a reduction solely catalyzed by the quinone oxidoreductase to produce the 2-keto carboxylic acid.

In some embodiments, the

is 3-hydroxy-propanal. In some embodiments, the 3-hydroxy-propanal is produced by dehydration of glycerol by a glycerol dehydratase enzyme exogenously expressed by the one or more non-naturally occurring microbial organisms.

In some embodiments, the method for producing the 2-keto carboxylic acid further comprises separating the 2-keto carboxylic acid from the one or more non-naturally occurring microbial organisms or a culture comprising the one or more non-naturally occurring microbial organisms.

In another aspect, provided herein is a method for producing 1,5-pentanediol, the method comprising

-   contacting pyruvate and 3-hydroxy-propanal with a hydratase-aldolase     and a quinone oxidoreductase to produce a 2-keto carboxylic acid of     formula:

wherein R is CH₂OH;

-   contacting the 2-keto carboxylic acid with a     2-keto-acid-decarboxylase to produce a 5-hydroxy-pentanal; and -   contacting the 5-hydroxy-pentanal with a primary alcohol     dehydrogenase to produce the 1,5-pentanediol, wherein the method is     performed in a culture comprising one or more non-naturally     occurring microbial organisms.

In another aspect, provided herein is a method for producing 1,5-pentanediol, the method comprising

-   contacting pyruvate and 3-hydroxy-propanal with a hydratase-aldolase     and a quinone oxidoreductase to produce a 2-keto carboxylic acid of     formula:

wherein R is CH₂OH;

-   contacting the 2-keto carboxylic acid with a     2-keto-acid-decarboxylase to produce a 5-hydroxy-pentanal; and -   contacting the 5-hydroxy-pentanal with a primary alcohol     dehydrogenase to produce the 1,5-pentanediol, wherein the method is     performed in a culture comprising two or more non-naturally     occurring microbial organisms.

In another aspect, provided herein is a method for producing 1,6-hexanediol, the method comprising

-   contacting pyruvate and 3-hydroxy-propanal with a hydratase-aldolase     and a quinone oxidoreductase to produce a 2-keto carboxylic acid of     formula:

wherein R is CH₂OH;

-   contacting the 2-keto carboxylic acid with a     6-hydroxy-2-oxohexanoate-2-reductase to produce     2,6-dihydroxy-hexanoate; -   contacting the 2,6-dihydroxy-hexanoate with a     2,6-dihydroxy-hexanoate CoA-transferase to produce     2,6-dihydroxy-hexanoyl-CoA; -   contacting the 2,6-dihydroxy-hexanoyl-CoA with a the     2,6-dihydroxy-hexanoyl-CoA 2-dehydratase to produce     6-hydroxy-2,3-dehydro-hexanoyl-CoA; -   contacting the 6-hydroxy-2,3-dehydro-hexanoyl-CoA with a     2,3-dehydro-hexanoyl-CoA 2,3-reductase to produce     6-hydroxy-hexanoyl-CoA; -   contacting the 6-hydroxy-hexanoyl-CoA with a 6-hydroxyhexanoyl-CoA     transferase to produce 6-hydroxy-hexanoate; -   contacting the 6-hydroxy-hexanoate with a 6-hydroxyhexanoate     1-reductase to produce 6-hydroxy-hexanal; and -   contacting the 6-hydroxy-hexanal with a 6-hydroxyhexanal 1-reductase     to produce the 1,6-hexanediol, wherein the method is performed in a     culture comprising one or more non-naturally occurring microbial     organisms.

In another aspect, provided herein is a method for producing 1,6-hexanediol, the method comprising

-   contacting pyruvate and 3-hydroxy-propanal with a hydratase-aldolase     and a quinone oxidoreductase to produce a 2-keto carboxylic acid of     formula:

wherein R is CH₂OH;

-   contacting the 2-keto carboxylic acid with a     6-hydroxy-2-oxohexanoate-2-reductase to produce     2,6-dihydroxy-hexanoate; -   contacting the 2,6-dihydroxy-hexanoate with a     2,6-dihydroxy-hexanoate CoA-transferase to produce     2,6-dihydroxy-hexanoyl-CoA; -   contacting the 2,6-dihydroxy-hexanoyl-CoA with a the     2,6-dihydroxy-hexanoyl-CoA 2-dehydratase to produce     6-hydroxy-2,3-dehydro-hexanoyl-CoA; -   contacting the 6-hydroxy-2,3-dehydro-hexanoyl-CoA with a     2,3-dehydro-hexanoyl-CoA 2,3-reductase to produce     6-hydroxy-hexanoyl-CoA; -   contacting the 6-hydroxy-hexanoyl-CoA with a 6-hydroxyhexanoyl-CoA     transferase to produce 6-hydroxy-hexanoate; -   contacting the 6-hydroxy-hexanoate with a 6-hydroxyhexanoate     1-reductase to produce 6-hydroxy-hexanal; and -   contacting the 6-hydroxy-hexanal with a 6-hydroxyhexanal 1-reductase     to produce the 1,6-hexanediol, wherein the method is performed in a     culture comprising two or more non-naturally occurring microbial     organisms.

In another aspect, provided herein is a method for producing 6-hydroxy-hexanoate, the method comprising

-   contacting pyruvate and 3-hydroxy-propanal with a hydratase-aldolase     and a quinone oxidoreductase to produce a 2-keto carboxylic acid of     formula:

wherein R is CH₂OH;

-   contacting the 2-keto carboxylic acid with a     6-hydroxy-2-oxohexanoate-2-reductase to produce     2,6-dihydroxy-hexanoate; -   contacting the 2,6-dihydroxy-hexanoate with a     2,6-dihydroxy-hexanoate CoA-transferase to produce     2,6-dihydroxy-hexanoyl-CoA; -   contacting the 2,6-dihydroxy-hexanoyl-CoA with a the     2,6-dihydroxy-hexanoyl-CoA 2-dehydratase to produce     6-hydroxy-2,3-dehydro-hexanoyl-CoA; -   contacting the 6-hydroxy-2,3-dehydro-hexanoyl-CoA with a     2,3-dehydro-hexanoyl-CoA 2,3-reductase to produce     6-hydroxy-hexanoyl-CoA; and -   contacting the 6-hydroxy-hexanoyl-CoA with a 6-hydroxyhexanoyl-CoA     transferase to produce the 6-hydroxy-hexanoate; -   wherein the method is performed in a culture comprising one or more     non-naturally occurring microbial organisms.

In another aspect, provided herein is a method for producing 6-hydroxy-hexanoate, the method comprising

-   contacting pyruvate and 3-hydroxy-propanal with a hydratase-aldolase     and a quinone oxidoreductase to produce a 2-keto carboxylic acid of     formula:

wherein R is CH₂OH;

-   contacting the 2-keto carboxylic acid with a     6-hydroxy-2-oxohexanoate-2-reductase to produce     2,6-dihydroxy-hexanoate; -   contacting the 2,6-dihydroxy-hexanoate with a     2,6-dihydroxy-hexanoate CoA-transferase to produce     2,6-dihydroxy-hexanoyl-CoA; -   contacting the 2,6-dihydroxy-hexanoyl-CoA with a the     2,6-dihydroxy-hexanoyl-CoA 2-dehydratase to produce     6-hydroxy-2,3-dehydro-hexanoyl-CoA; -   contacting the 6-hydroxy-2,3-dehydro-hexanoyl-CoA with a     2,3-dehydro-hexanoyl-CoA 2,3-reductase to produce     6-hydroxy-hexanoyl-CoA; and -   contacting the 6-hydroxy-hexanoyl-CoA with a 6-hydroxyhexanoyl-CoA     transferase to produce the 6-hydroxy-hexanoate;     wherein the method is performed in a culture comprising two or more     non-naturally occurring microbial organisms.

In another aspect, provided herein is a method for producing adipic acid (AA), the method comprising

-   contacting pyruvate and 3-hydroxy-propanal with a hydratase-aldolase     and a quinone oxidoreductase to produce a 2-keto carboxylic acid of     formula:

wherein R is CH₂OH;

-   contacting the 2-keto carboxylic acid with a     6-hydroxy-2-oxohexanoate-2-reductase to produce     2,6-dihydroxy-hexanoate; -   contacting the 2,6-dihydroxy-hexanoate with a     2,6-dihydroxy-hexanoate CoA-transferase to produce     2,6-dihydroxy-hexanoyl-CoA; -   contacting the 2,6-dihydroxy-hexanoyl-CoA with a the     2,6-dihydroxy-hexanoyl-CoA 2-dehydratase to produce     6-hydroxy-2,3-dehydro-hexanoyl-CoA; -   contacting the 6-hydroxy-2,3-dehydro-hexanoyl-CoA with a     2,3-dehydro-hexanoyl-CoA 2,3-reductase to produce     6-hydroxy-hexanoyl-CoA; -   contacting the 6-hydroxy-hexanoyl-CoA with a 6-hydroxyhexanoyl-CoA     transferase to produce 6-hydroxy-hexanoate; -   contacting the 6-hydroxy-hexanoate with a 6-hydroxyhexanoate     dehydrogenase to produce 6-oxo-hexanoate; and -   contacting the 6-oxo-hexanoate with a 6-oxo-hexanoate oxidase to     produce the adipic acid, -   wherein the method is performed in a culture comprising one or more     non-naturally occurring microbial organisms.

In another aspect, provided herein is a method for producing adipic acid (AA), the method comprising

-   contacting pyruvate and 3-hydroxy-propanal with a hydratase-aldolase     and a quinone oxidoreductase to produce a 2-keto carboxylic acid of     formula:

wherein R is CH₂OH;

-   contacting the 2-keto carboxylic acid with a     6-hydroxy-2-oxohexanoate-2-reductase to produce     2,6-dihydroxy-hexanoate; -   contacting the 2,6-dihydroxy-hexanoate with a     2,6-dihydroxy-hexanoate CoA-transferase to produce     2,6-dihydroxy-hexanoyl-CoA; -   contacting the 2,6-dihydroxy-hexanoyl-CoA with a the     2,6-dihydroxy-hexanoyl-CoA 2-dehydratase to produce     6-hydroxy-2,3-dehydro-hexanoyl-CoA; -   contacting the 6-hydroxy-2,3-dehydro-hexanoyl-CoA with a     2,3-dehydro-hexanoyl-CoA 2,3-reductase to produce     6-hydroxy-hexanoyl-CoA; -   contacting the 6-hydroxy-hexanoyl-CoA with a 6-hydroxyhexanoyl-CoA     transferase to produce 6-hydroxy-hexanoate; -   contacting the 6-hydroxy-hexanoate with a 6-hydroxyhexanoate     dehydrogenase to produce 6-oxo-hexanoate; and -   contacting the 6-oxo-hexanoate with a 6-oxo-hexanoate oxidase to     produce the adipic acid, -   wherein the method is performed in a culture comprising two or more     non-naturally occurring microbial organisms.

In some embodiments, the hydratase-aldolase is an enzyme having an EC number 4.1.2.45 or EC number 4.1.2.34 or EC number 4.1.1.4. In some embodiments, the hydratase-aldolase is an enzyme having an EC number 4.1.2.45. In some embodiments, the hydratase-aldolase is a trans-o-hydroxybenzylidenepyruvate hydratase-aldolase having an EC number 4.1.2.45. In some embodiments, the hydratase-aldolase is an enzyme having an EC number 4.1.2.34. In some embodiments, the hydratase-aldolase is an enzyme having an EC number 4.1.1.4.

In some embodiments, the hydratase-aldolase is an enzyme selected from the group of enzymes identified under Genbank or RefSeq or Uniprot ID Nos. D7C0E5, P0A144, Q79EM8, A0A0N0AHI8, A0A0N1FRY3, M3DYR1, W7SU48, A0A286PH18, Q9X9Q6, Q9WXH7, A4XDS1, F2J6N9, A0A063BFL5, Q9ZHH6, A0A0C1K853, WP_034398482, PYK12191, WP_115478033, WP_028222253, WP_013654807, WP_059403060, WP_092508530, WP_116642627, WP_009770659, WP_107818191, WP_003292061, PYN48855, WP_122212965, WP_028217297, WP_034507049, KMK64081.1, WP_070028041.1, or KZL92449.1. In some embodiments, the hydratase-aldolase is an enzyme selected from the group of enzymes identified under GenBank, RefSeq, or Uniprot ID Nos. D7C0E5, P0A144, Q79EM8, A0A0N0AHI8, A0A0N1FRY3, M3DYR1, W7SU48, A0A286PH18, Q9X9Q6, Q9WXH7, A4XDS1, F2J6N9, A0A063BFL5, Q9ZHH6, A0A0C1K853, WP_034398482, PYK12191, A0A370X7D8, WP_028222253, F2J6L6, A0A0N0L9F6, A0A1G9YWG7, A0A2U1BT09, A0A244DHE8, WP_107818191, A0A023WZF9, PYN48855, A0A421PAQ6, WP_028217297, WP_034507049, KMK64081.1, WP_070028041.1, or KZL92449.1. In some embodiments, the hydratase-aldolase is an enzyme comprising a sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:84, SEQ ID NO:85, or SEQ ID NO:86.

In some embodiments, the hydratase-aldolase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme selected from the group of enzymes identified under Genbank or RefSeq or Uniprot ID Nos. D7C0E5, P0A144, Q79EM8, A0A0N0AHI8, A0A0N1FRY3, M3DYR1, W7SU48, A0A286PH18, Q9X9Q6, Q9WXH7, A4XDS1, F2J6N9, A0A063BFL5, Q9ZHH6, A0A0C1K853, WP_034398482, PYK12191, A0A370X7D8, WP_028222253, F2J6L6, A0A0N0L9F6, A0A1G9YWG7, A0A2U1BT09, A0A244DHE8, WP_107818191, A0A023WZF9, PYN48855, A0A421PAQ6, WP_028217297, WP_034507049, KMK64081.1, WP_070028041.1, or KZL92449.1. In some embodiments, the hydratase-aldolase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme comprising a sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:84, SEQ ID NO:85, or SEQ ID NO:86.

In some embodiments, the hydratase-aldolase is an enzyme selected from Tables 1, 5, 6, 7, and 8. In some embodiments, the hydratase-aldolase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme selected from Tables 1, 5, 6, 7, and 8.

In some embodiments, the hydratase-aldolase further comprises one or more protein tags. In some embodiments, the protein tags are selected from polyhistidine tag, a GST tag (glutathione-S-transferase tag), a HA tag (hemagglutinin tag), a FLAG tag, a Myc tag, a maltose binding protein tag, a chitin binding protein tag, and a fluorescent tag.

In some embodiments, the quinone oxidoreductase is an enzyme having an EC number 1.6.5. In some embodiments, the quinone oxidoreductase is an enzyme having an EC number 1.6.5.5. In some embodiments, the quinone oxidoreductase is an enzyme selected from the group of enzymes identified under Under GenBank, RefSeq, or Uniprot ID Nos. P28304, P40783, Q0K2I0, A0A1Z1SRY9, P43903, I7G8G0, or Q142L2, ALK19324.1, A0A1G9R408, G4Q8R5, ANA98723.1, K0EUQ3, A0A061CRS8, Q9A212, A0A1I6RWW2, WP_026197277.1, Q5NKZ3, WP_012333034.1, or WP_136898000.1. In some embodiments, the quinone oxidoreductase is an enzyme comprising a sequence of SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, or SEQ ID NO:97.

In some embodiments, the quinone oxidoreductase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme selected from the group of enzymes identified under Under GenBank, RefSeq, or Uniprot ID Nos. P28304, P40783, Q0K2I0, A0A1Z1SRY9, P43903, I7G8G0, or Q142L2, ALK19324.1, A0A1G9R408, G4Q8R5, ANA98723.1, K0EUQ3, A0A061CRS8, Q9A212, A0A1I6RWW2, WP_026197277.1, Q5NKZ3, WP_012333034.1, or WP_136898000.1. In some embodiments, the quinone oxidoreductase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme comprising a sequence of SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, or SEQ ID NO:97.

In some embodiments, the quinone oxidoreductase further comprises one or more protein tags. In some embodiments, the protein tags are selected from polyhistidine tag, a GST tag (glutathione-S-transferase tag), a HA tag (hemagglutinin tag), a FLAG tag, a Myc tag, a maltose binding protein tag, a chitin binding protein tag, and a fluorescent tag.

In some embodiments, at least one of the hydratase-aldolase and the quinone oxidoreductase is exogenously expressed by the one or more non-naturally occurring microbial organisms. In some embodiments, at least one of the hydratase-aldolase and the quinone oxidoreductase is exogenously expressed by the two or more non-naturally occurring microbial organisms.

In some embodiments, at least one of the hydratase-aldolase and the quinone oxidoreductase enzymes are expressed by one or more exogenous genes expressed by the one or more non-naturally occurring microorganisms. In some embodiments, at least one of the hydratase-aldolase and the quinone oxidoreductase enzymes are expressed by one or more exogenous genes expressed by the two or more non-naturally occurring microorganisms. In some embodiments, at least one of the hydratase-aldolase and the quinone oxidoreductase enzymes are expressed by two or more exogenous genes expressed by the one or more non-naturally occurring microorganisms. In some embodiments, at least one of the hydratase-aldolase and the quinone oxidoreductase enzymes are expressed by two or more exogenous genes expressed by the two or more non-naturally occurring microorganisms. One or more exogenous genes includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, or more, exogenous genes. Two or more exogenous genes includes 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, or more, exogenous genes.

In some embodiments, the hydratase-aldolase is exogenously expressed by the one or more non-naturally occurring microbial organisms. In some embodiments, the hydratase-aldolase is exogenously expressed by the two or more non-naturally occurring microbial organisms.

In some embodiments, the quinone oxidoreductase is exogenously expressed by the one or more non-naturally occurring microbial organisms. In some embodiments, the quinone oxidoreductase is overexpressed by the one or more non-naturally occurring microbial organisms. In some embodiments, the quinone oxidoreductase is exogenously expressed by the two or more non-naturally occurring microbial organisms. In some embodiments, the quinone oxidoreductase is overexpressed by the two or more non-naturally occurring microbial organisms.

In some embodiments, the hydratase-aldolase is exogenously expressed by the one or more non-naturally occurring microbial organisms and the quinone oxidoreductase is overexpressed by the one or more non-naturally occurring microbial organisms. In some embodiments, the hydratase-aldolase is exogenously expressed by the two or more non-naturally occurring microbial organisms and the quinone oxidoreductase is overexpressed by the two or more non-naturally occurring microbial organisms.

In some embodiments, the 2-keto-acid-decarboxylase and the primary alcohol dehydrogenase are expressed by the one or more non-naturally occurring microbial organisms. In some embodiments, the 2-keto-acid-decarboxylase and the primary alcohol dehydrogenase are exogenously expressed by the one or more non-naturally occurring microbial organisms.

In some embodiments, the 2-keto-acid-decarboxylase is an enzyme selected from the group of enzymes identified under EC number 4.1.1.1; EC number 4.1.1.2; EC number 4.1.1.3; EC number 4.1.1.4; EC number 4.1.1.5; EC number 4.1.1.6; EC number 4.1.1.7; EC number 4.1.1.11; EC number 4.1.1.12; EC number 4.1.1.15; EC number 4.1.1.16; EC number 4.1.1.17; EC number 4.1.1.18; EC number 4.1.1.19; EC number 4.1.1.20; EC number 4.1.1.34; EC number 4.1.1.35; EC number 4.1.1.40; EC number 4.1.1.54; EC number 4.1.1.56; EC number 4.1.1.71; EC number 4.1.1.72; EC number 4.1.1.73; EC number 4.1.1.74; EC number 4.1.1.75; or EC number 4.1.1.77. In some embodiments, the 2-keto-acid-decarboxylase is an enzyme selected from the group of enzymes identified under Uniprot ID No. Q6QBS4, A7M7D6, or P20906. In some embodiments, the 2-keto-acid-decarboxylase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme selected from the group of enzymes identified under Uniprot ID Nos. Q6QBS4, A7M7D6, or P20906.

In some embodiments, the 2-keto-acid-decarboxylase further comprises one or more protein tags. In some embodiments, the protein tags are selected from polyhistidine tag, a GST tag (glutathione-S-transferase tag), a HA tag (hemagglutinin tag), a FLAG tag, a Myc tag, a maltose binding protein tag, a chitin binding protein tag, and a fluorescent tag.

In some embodiments, the primary alcohol dehydrogenase is an enzyme having an EC number 1.1.1.61. In some embodiments, the primary alcohol dehydrogenase is an enzyme selected from the group of enzymes identified under Uniprot or GenBank ID Nos. NP_417279.1, NP_349892.1, NP_349891.1, BAB12273.1, L21902.1, Q94B07, AAB03015.1, NP_014032.1, NP_013892.1, NP_015019.1, NP_010996.2, ABX39192.1, XP_001210625.1, ABO67118, ABO68223, BAE77068.1, or CAA47743.1. In some embodiments, the primary alcohol dehydrogenase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme selected from the group of enzymes identified under Uniprot or GenBank ID Nos. NP_417279.1, NP_349892.1, NP_349891.1, BAB12273.1, L21902.1, Q94B07, AAB03015.1, NP_014032.1, NP_013892.1, NP_015019.1, NP_010996.2, ABX39192.1, XP_001210625.1, ABO67118, ABO68223, BAE77068.1, or CAA47743.1. In some embodiments, the primary alcohol dehydrogenase is an enzyme comprising a sequence of SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, or SEQ ID NO:74. In some embodiments, the primary alcohol dehydrogenase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme comprising a sequence of SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, or SEQ ID NO:74.

In some embodiments, the primary alcohol dehydrogenase further comprises one or more protein tags. In some embodiments, the protein tags are selected from polyhistidine tag, a GST tag (glutathione-S-transferase tag), a HA tag (hemagglutinin tag), a FLAG tag, a Myc tag, a maltose binding protein tag, a chitin binding protein tag, and a fluorescent tag.

In some embodiments, the hydratase-aldolase is an enzyme identified under Uniprot ID No. A0A286PH18; the quinone oxidoreductase is an enzyme identified under Uniprot ID No. P28304; the 2-keto-acid-decarboxylase is an enzyme identified under Uniprot ID No. Q6QBS4; and the primary alcohol dehydrogenase is an enzyme identified under Uniprot or GenBank ID Nos. D6Z860, YP_001705436.1, ANO06407.1, AAR91681.1, AHH98121.1, ANB00612.1, ANO04655.1, A0R484, AFP42026.1, GAJ86510.1, YP_001704097.1, ANA99315.1, GAJ83027.1, ANA98925.1, ANA98924.1, ANO04656.1, YP_001703694. In some embodiments, the hydratase-aldolase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme identified under Uniprot ID No. A0A286PH18; the quinone oxidoreductase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme identified under Uniprot ID No. P28304; the 2-keto-acid-decarboxylase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme identified under Uniprot ID No. Q6QBS4; and the primary alcohol dehydrogenase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme identified under Uniprot or GenBank ID Nos. D6Z860, YP_001705436.1, ANO06407.1, AAR91681.1, AHH98121.1, ANB00612.1, ANO04655.1, A0R484, AFP42026.1, GAJ86510.1, YP_001704097.1, ANA99315.1, GAJ83027.1, ANA98925.1, ANA98924.1, ANO04656.1, YP_001703694.

In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase, the 2,6-dihydroxy-hexanoate CoA-transferase, the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase, the 2,3-dehydro-hexanoyl-CoA 2,3-reductase, the 6-hydroxyhexanoyl-CoA transferase, the 6-hydroxyhexanoate 1-reductase, and the 6-hydroxyhexanal 1-reductase are expressed by the one or more non-naturally occurring microbial organisms.

In some embodiments, wherein the 6-hydroxy-2-oxohexanoate-2-reductase, the 2,6-dihydroxy-hexanoate CoA-transferase, the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase, the 2,3-dehydro-hexanoyl-CoA 2,3-reductase, the 6-hydroxyhexanoyl-CoA transferase, the 6-hydroxyhexanoate 1-reductase, and the 6-hydroxyhexanal 1-reductase are exogenously expressed by the one or more non-naturally occurring microbial organisms.

In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase is an selected from the group of enzymes identified under an EC number 1.1.99.6, EC number 1.1.1.169, EC number 1.1.1.215, EC number 1.1.1.28, or EC number 1.1.1.110; the 2,6-dihydroxy-hexanoate CoA-transferase is an enzyme having an EC number 2.8.3, EC number 2.8.3.1, or EC number 2.8.3.12; the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase is an enzyme having an EC number 4.2.1.167; the 2,3-dehydro-hexanoyl-CoA 2,3-reductase is an enzyme having an EC number 1.3.1.44; the 6-hydroxyhexanoyl-CoA transferase is an enzyme having an EC number 2.8.3, EC number 2.8.3.1, or EC number 2.8.3.12; the 6-hydroxyhexanoate 1-reductase is an enzyme having an EC number 1.2.99.6; and the 6-hydroxyhexanal 1-reductase is an enzyme having an EC number 1.1.1.

In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase is an enzyme selected from the group of enzymes identified under Uniprot or GenBank ID Nos. WP_003431407.1, BAL51292.1, Q5FTU6, AKC64094.1, WP_002876862.1, AGP69017.1, WP_003640741.1, AKC6409; the 2,6-dihydroxy-hexanoate CoA-transferase is an enzyme identified under Uniprot ID No. T4VW93; the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase is an enzyme identified under Uniprot ID Nos. Q5U924, Q5U925, and Q5U923; the 2,3-dehydro-hexanoyl-CoA 2,3-reductase is an enzyme identified under Uniprot ID No. Q73Q47; the 6-hydroxyhexanoyl-CoA transferase is an enzyme identified under Uniprot ID No. T4VW93; the 6-hydroxyhexanoate 1-reductase is an enzyme identified under Uniprot or GenBank ID Nos. D6Z860, YP_001705436.1, ANO06407.1, AAR91681.1, AHH-98121.1, ANB00612.1, ANO04655.1, A0R484, AFP42026.1, GAJ86510.1, YP_001704097.1, ANA99315.1, GAJ83027.1, ANA98925.1, ANA98924.1, ANO04656.1, YP_001703694.1, WP_036338301.1, WP_007472106.1, or A0QWI7; and the 6-hydroxyhexanal 1-reductase is an enzyme identified under Uniprot or GenBank ID Nos. D6Z860, YP_001705436.1, ANO06407.1, AAR91681.1, AHH-98121.1, ANB00612.1, ANO04655.1, A0R484, AFP42026.1, GAJ86510.1, YP_001704097.1, ANA99315.1, GAJ83027.1, ANA98925.1, ANA98924.1, ANO04656.1, YP_001703694.

In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase is an enzyme selected from the group of enzymes identified under Uniprot or GenBank ID Nos. WP_003431407.1, BAL51292.1, Q5FTU6, AKC64094.1, WP_002876862.1, AGP69017.1, WP_003640741.1, AKC6409; the 2,6-dihydroxy-hexanoate CoA-transferase is an enzyme identified under Uniprot ID Nos. T4VW93, A0A0C7GD16, A0A175L1W4, or 0A2X3BTQ9; the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase is an enzyme identified under Uniprot ID Nos. Q5U924, Q5U925, and Q5U923; or A0A2X3BK09, A0A2X3BU19, and A0A1V9IXA9; the 2,3-dehydro-hexanoyl-CoA 2,3-reductase is an enzyme identified under Uniprot ID No. Q73Q47; the 6-hydroxyhexanoyl-CoA transferase is an enzyme identified under Uniprot ID No. T4VW93, A0A0C7GD16, A0A175L1W4, or A0A2X3BTQ9; the 6-hydroxyhexanoate 1-reductase is an enzyme identified under Uniprot or GenBank ID Nos. D6Z860, YP_001705436.1, ANO06407.1, AAR91681.1, AHH-98121.1, ANB00612.1, ANO04655.1, A0R484, AFP42026.1, GAJ86510.1, YP_001704097.1, ANA99315.1, GAJ83027.1, ANA98925.1, ANA98924.1, ANO04656.1, YP_001703694.1, WP_036338301.1, WP_007472106.1, or A0QWI7; and the 6-hydroxyhexanal 1-reductase is an enzyme identified under Uniprot or GenBank ID Nos. D6Z860, YP_001705436.1, ANO06407.1, AAR91681.1, AHH-98121.1, ANB00612.1, ANO04655.1, A0R484, AFP42026.1, GAJ86510.1, YP_001704097.1, ANA99315.1, GAJ83027.1, ANA98925.1, ANA98924.1, ANO04656.1, YP_001703694.

In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme identified under Uniprot or GenBank ID Nos. WP_003431407.1, BAL51292.1, Q5FTU6, AKC64094.1, WP_002876862.1, AGP69017.1, WP_003640741.1, AKC64095.1, and AKC64094.1; the 2,6-dihydroxy-hexanoate CoA-transferase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme identified under Uniprot ID Nos. T4VW93, A0A0C7GD16, A0A175L1W4, or 0A2X3BTQ9; the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme identified under Uniprot ID Nos. Q5U924, Q5U925, and Q5U923; or A0A2X3BK09, A0A2X3BU19, and A0A1V9IXA9; the 2,3-dehydro-hexanoyl-CoA 2,3-reductase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme identified under Uniprot ID No. Q73Q47; the 6-hydroxyhexanoyl-CoA transferase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme identified under Uniprot ID No. T4VW93, A0A0C7GD16, A0A175L1W4, or A0A2X3BTQ9; the 6-hydroxyhexanoate 1-reductase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme identified under Uniprot or GenBank ID Nos. D6Z860, YP_001705436.1, ANO06407.1, AAR91681.1, AHH-98121.1, ANB00612.1, ANO04655.1, A0R484, AFP42026.1, GAJ86510.1, YP_001704097.1, ANA99315.1, GAJ83027.1, ANA98925.1, ANA98924.1, ANO04656.1, YP_001703694.1, WP_036338301.1, WP_007472106.1, or A0QWI7; and the 6-hydroxyhexanal 1-reductase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme identified under Uniprot or GenBank ID Nos. D6Z860, YP_001705436.1, ANO06407.1, AAR91681.1, AHH98121.1, ANB00612.1, ANO04655.1, A0R484, AFP42026.1, GAJ86510.1, YP_001704097.1, ANA99315.1, GAJ83027.1, ANA98925.1, ANA98924.1, ANO04656.1, YP001703694.

In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase is an enzyme comprising a sequence of SEQ ID NO:53, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, or SEQ ID NO:105; the 2,6-dihydroxy-hexanoate CoA-transferase is an enzyme comprising a sequence of SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, or SEQ ID NO:58; the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase is an enzyme comprising a sequence of SEQ ID NO:59, SEQ ID NO:61, and SEQ ID NO:63; or SEQ ID NO:60, SEQ ID NO:62, and SEQ ID NO:64; the 2,3-dehydro-hexanoyl-CoA 2,3-reductase is an enzyme comprising a sequence of SEQ ID NO:65; the 6-hydroxyhexanoyl-CoA transferase is an enzyme comprising a sequence of SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, or SEQ ID NO:58; the 6-hydroxyhexanoate 1-reductase is an enzyme comprising a sequence of SEQ ID NO:66, SEQ ID NO:67, or SEQ ID NO:68; and the 6-hydroxyhexanal 1-reductase is an enzyme comprising a sequence of SEQ ID NO:70.

In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme comprising a sequence of SEQ ID NO:53, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, or SEQ ID NO:105; the 2,6-dihydroxy-hexanoate CoA-transferase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme comprising a sequence of SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, or SEQ ID NO:58; the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme comprising a sequence of SEQ ID NO:59, SEQ ID NO:61, and SEQ ID NO:63; or SEQ ID NO:60, SEQ ID NO:62, and SEQ ID NO:64; the 2,3-dehydro-hexanoyl-CoA 2,3-reductase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme comprising a sequence of SEQ ID NO:65; the 6-hydroxyhexanoyl-CoA transferase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme comprising a sequence of SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, or SEQ ID NO:58; the 6-hydroxyhexanoate 1-reductase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme comprising a sequence of SEQ ID NO:66, SEQ ID NO:67, or SEQ ID NO:68; and the 6-hydroxyhexanal 1-reductase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme comprising a sequence of SEQ ID NO:70.

In some embodiments, one or more of the 6-hydroxy-2-oxohexanoate-2-reductase, the 2,6-dihydroxy-hexanoate CoA-transferase, the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase, the 2,3-dehydro-hexanoyl-CoA 2,3-reductase, the 6-hydroxyhexanoyl-CoA transferase, the 6-hydroxyhexanoate 1-reductase, and the 6-hydroxyhexanal 1-reductase further comprise one or more protein tags. In some embodiments, the protein tags are selected from polyhistidine tag, a GST tag (glutathione-S-transferase tag), a HA tag (hemagglutinin tag), a FLAG tag, a Myc tag, a maltose binding protein tag, a chitin binding protein tag, and a fluorescent tag.

In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase is an selected from the group of enzymes identified under an EC number 1.1.99.6, EC number 1.1.1.169, EC number 1.1.1.215, EC number 1.1.1.28, or EC number 1.1.1.110; the 2,6-dihydroxy-hexanoate CoA-transferase is an enzyme having an EC number 2.8.3, EC number 2.8.3.1, or EC number 2.8.3.12; the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase is an enzyme having an EC number 4.2.1.167; the 2,3-dehydro-hexanoyl-CoA 2,3-reductase is an enzyme having an EC number 1.3.1.44; and the 6-hydroxyhexanoyl-CoA transferase is an enzyme having an EC number 2.8.3, EC number 2.8.3.1, or EC number 2.8.3.12.

In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase is an enzyme identified under Uniprot or GenBank ID Nos. WP_003431407.1, BAL51292.1, Q5FTU6, AKC64094.1, WP_002876862.1, AGP69017.1, WP_003640741.1, AKC64095.1, and AKC64094.1; the 2,6-dihydroxy-hexanoate CoA-transferase is an enzyme identified under Uniprot ID Nos. T4VW93, A0A2X3BTQ9, A0A0C7GD16, or A0A175L1W4; the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase is an enzyme identified under Uniprot ID Nos. Q5U924, Q5U925, and Q5U923; or A0A2X3BK09, A0A2X3BU19, and A0A1V9IXA9; the 2,3-dehydro-hexanoyl-CoA 2,3-reductase is an enzyme identified under Uniprot ID No. Q73Q47; and the 6-hydroxyhexanoyl-CoA transferase is an enzyme identified under Uniprot ID Nos. T4VW93, A0A2X3BTQ9, A0A0C7GD16, or A0A175L1W4.

In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme identified under Uniprot or GenBank ID Nos. WP_003431407.1, BAL51292.1, Q5FTU6, AKC64094.1, WP_002876862.1, AGP69017.1, WP_003640741.1, AKC64095.1, and AKC64094.1; the 2,6-dihydroxy-hexanoate CoA-transferase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme identified under Uniprot ID Nos. T4VW93, A0A2X3BTQ9, A0A0C7GD16, or A0A175L1W4; the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme identified under Uniprot ID Nos. Q5U924, Q5U925, and Q5U923; or A0A2X3BK09, A0A2X3BU19, and A0A1V9IXA9; the 2,3-dehydro-hexanoyl-CoA 2,3-reductase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme identified under Uniprot ID No. Q73Q47; and the 6-hydroxyhexanoyl-CoA transferase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme identified under Uniprot ID Nos. T4VW93, A0A2X3BTQ9, A0A0C7GD16, or A0A175L1W4.

In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase is an enzyme comprising a sequence of SEQ ID NO:53, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, or SEQ ID NO:105; the 2,6-dihydroxy-hexanoate CoA-transferase is an enzyme comprising a sequence of SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, or SEQ ID NO:58; the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase is an enzyme comprising a sequence of SEQ ID NO:59, SEQ ID NO:61, and SEQ ID NO:63; or SEQ ID NO:60, SEQ ID NO:62, and SEQ ID NO:64; the 2,3-dehydro-hexanoyl-CoA 2,3-reductase is an enzyme comprising a sequence of SEQ ID NO:65; and the 6-hydroxyhexanoyl-CoA transferase is an enzyme comprising a sequence of SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, or SEQ ID NO:58.

In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme comprising a sequence of SEQ ID NO:53, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, or SEQ ID NO:105; the 2,6-dihydroxy-hexanoate CoA-transferase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme comprising a sequence of SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, or SEQ ID NO:58; the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme comprising a sequence of SEQ ID NO:59, SEQ ID NO:61, and SEQ ID NO:63; or SEQ ID NO:60, SEQ ID NO:62, and SEQ ID NO:64; the 2,3-dehydro-hexanoyl-CoA 2,3-reductase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme comprising a sequence of SEQ ID NO:65; and the 6-hydroxyhexanoyl-CoA transferase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme comprising a sequence of SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, or SEQ ID NO:58.

In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase is an selected from the group of enzymes identified under an EC number 1.1.99.6, EC number 1.1.1.169, EC number 1.1.1.215, EC number 1.1.1.28, or EC number 1.1.1.110; the 2,6-dihydroxy-hexanoate CoA-transferase is an enzyme having an EC number 2.8.3, EC number 2.8.3.1, or EC number 2.8.3.12; the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase is an enzyme having an EC number 4.2.1.167; the 2,3-dehydro-hexanoyl-CoA 2,3-reductase is an enzyme having an EC number 1.3.1.44; the 6-hydroxyhexanoyl-CoA transferase is an enzyme having an EC number 2.8.3, EC number 2.8.3.1, or EC number 2.8.3.12; the 6-hydroxyhexanoate dehydrogenase is an enzyme having an EC number 1.1.1.258; and the 6-oxo-hexanoate oxidase is an enzyme having an EC number 1.2.1.63.

In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase is an enzyme identified under Uniprot ID No. Q5FTU6; the 2,6-dihydroxy-hexanoate CoA-transferase is an enzyme identified under Uniprot ID Nos. T4VW93 or A0A2X3BTQ9; the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase is an enzyme identified under Uniprot ID Nos. Q5U924, Q5U925, and Q5U923; or A0A2X3BK09, A0A2X3BU19, and A0A1V9IXA9; the 2,3-dehydro-hexanoyl-CoA 2,3-reductase is an enzyme identified under Uniprot ID No. Q73Q47; the 6-hydroxyhexanoyl-CoA transferase is an enzyme identified under Uniprot ID Nos. T4VW93 or A0A2X3BTQ9; the 6-hydroxyhexanoate dehydrogenase is an enzyme identified under Uniprot ID Nos. Q7WVD0 or Q84H78; and the 6-oxo-hexanoate oxidase is an enzyme identified under Uniprot ID No. Q9R2F4.

In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme identified under Uniprot ID No. Q5FTU6; the 2,6-dihydroxy-hexanoate CoA-transferase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme identified under Uniprot ID Nos. T4VW93 or A0A2X3BTQ9; the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme identified under Uniprot ID Nos. Q5U924, Q5U925, and Q5U923; or A0A2X3BK09, A0A2X3BU19, and A0A1V9IXA9; the 2,3-dehydro-hexanoyl-CoA 2,3-reductase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme identified under Uniprot ID No. Q73Q47; the 6-hydroxyhexanoyl-CoA transferase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme identified under Uniprot ID Nos. T4VW93 or A0A2X3BTQ9; the 6-hydroxyhexanoate dehydrogenase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme identified under Uniprot ID Nos. Q7WVD0 or Q84H78; and the 6-oxo-hexanoate oxidase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme identified under Uniprot ID No. Q9R2F4.

In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase is an enzyme comprising a sequence of SEQ ID NO:53; the 2,6-dihydroxy-hexanoate CoA-transferase is an enzyme comprising a sequence of SEQ ID NO:55 or SEQ ID NO:58; the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase is an enzyme comprising a sequence of SEQ ID NO:59, SEQ ID NO:61, and SEQ ID NO:63; or SEQ ID NO:60, SEQ ID NO:62, and SEQ ID NO:64; the 2,3-dehydro-hexanoyl-CoA 2,3-reductase is an enzyme comprising a sequence of SEQ ID NO:65; the 6-hydroxyhexanoyl-CoA transferase is an enzyme comprising a sequence of SEQ ID NO:55 or SEQ ID NO:58; the 6-hydroxyhexanoate dehydrogenase is an enzyme identified comprising a sequence of SEQ ID NO:71 or SEQ ID NO:72; and the 6-oxo-hexanoate oxidase is an enzyme comprising a sequence of SEQ ID NO:75.

In some embodiments, the 6-hydroxy-2-oxohexanoate-2-reductase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme comprising a sequence of SEQ ID NO:53; the 2,6-dihydroxy-hexanoate CoA-transferase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme comprising a sequence of SEQ ID NO:55 or SEQ ID NO:58; the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme comprising a sequence of SEQ ID NO:59, SEQ ID NO:61, and SEQ ID NO:63; or SEQ ID NO:60, SEQ ID NO:62, and SEQ ID NO:64; the 2,3-dehydro-hexanoyl-CoA 2,3-reductase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme comprising a sequence of SEQ ID NO:65; the 6-hydroxyhexanoyl-CoA transferase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme comprising a sequence of SEQ ID NO:55 or SEQ ID NO:58; the 6-hydroxyhexanoate dehydrogenase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme identified comprising a sequence of SEQ ID NO:71 and SEQ ID NO:72; and the 6-oxo-hexanoate oxidase has at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, or more, to an enzyme comprising a sequence of SEQ ID NO:75.

In some embodiments, one or more of the 6-hydroxy-2-oxohexanoate-2-reductase, the 2,6-dihydroxy-hexanoate CoA-transferase, the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase, the 2,3-dehydro-hexanoyl-CoA 2,3-reductase, the 6-hydroxyhexanoyl-CoA transferase, 6-hydroxyhexanoate dehydrogenase, and the 6-oxo-hexanoate oxidase further comprise one or more protein tags. In some embodiments, the protein tags are selected from polyhistidine tag, a GST tag (glutathione-S-transferase tag), a HA tag (hemagglutinin tag), a FLAG tag, a Myc tag, a maltose binding protein tag, a chitin binding protein tag, and a fluorescent tag

In some embodiments, the pyruvate is produced from carbon sources selected from glycerol, glucose, xylose, arabinose, galactose, mannose, fructose, sucrose, and starch, or a combination of thereof.

In some embodiments, the 3-hydroxy-propanal is produced by dehydration of glycerol by a glycerol dehydratase enzyme exogenously expressed by the one or more non-naturally occurring microbial organisms.

The one or more non-naturally occurring microbial organisms include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, or more non-naturally occurring microbial organisms. The two or more non-naturally occurring microbial organisms include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, or more non-naturally occurring microbial organisms. In some embodiments, the method disclosed herein is performed in the presence of one non-naturally occurring microbial organism. In some embodiments, the method disclosed herein is performed in the presence of two non-naturally occurring microbial organisms. In some embodiments, the method disclosed herein is performed in the presence of three non-naturally occurring microbial organisms. In some embodiments, the method disclosed herein is performed in the presence of four non-naturally occurring microbial organisms. In some embodiments, the method disclosed herein is performed in the presence of five non-naturally occurring microbial organisms.

Throughout this application various publications have been referenced. The disclosure of these publications in their entireties, including GenBank accession number(s) or Uniprot ID number(s) or RefSeq ID numbers in these publications, are hereby incorporated by reference in this application in order to more fully describe the state of the art to which this present disclosure pertains.

In some embodiments, the present disclosure provides the following Embodiments as examples:

1. A method for producing a 2-keto carboxylic acid of formula:

wherein R is H, CH₃, or CH₂OH; the method comprising contacting pyruvate and

with a hydratase-aldolase and a quinone oxidoreductase in a culture comprising one or more non-naturally occurring microbial organisms to produce the 2-keto carboxylic acid; wherein the hydratase-aldolase and the quinone oxidoreductase are expressed by the one or more non-naturally occurring microbial organisms. 2. The method of Embodiment 1, wherein at least one of the hydratase-aldolase and the quinone oxidoreductase is exogenously expressed by the one or more non-naturally occurring microbial organisms. 3. The method of Embodiment 1, wherein the hydratase-aldolase is exogenously expressed by the one or more non-naturally occurring microbial organisms. 4. The method of Embodiment 1, wherein the quinone oxidoreductase is exogenously expressed by the one or more non-naturally occurring microbial organisms. 5. The method of Embodiment 1, wherein the quinone oxidoreductase is overexpressed by the one or more non-naturally occurring microbial organisms. 6. The method of Embodiment 1, wherein the hydratase-aldolase is exogenously expressed by the one or more non-naturally occurring microbial organisms and the quinone oxidoreductase is overexpressed by the one or more non-naturally occurring microbial organisms. 7. The method of any one of Embodiments 1-6, wherein

is 3-hydroxy-propanal. 8. The method of Embodiment 7, wherein the 3-hydroxy-propanal is produced by dehydration of glycerol by a glycerol dehydratase enzyme exogenously expressed by the one or more non-naturally occurring microbial organisms. 9. The method of any one of Embodiments 1-8, further comprising separating the 2-keto carboxylic acid from the one or more non-naturally occurring microbial organisms or a culture comprising the one or more non-naturally occurring microbial organisms. 10. A method for producing a 2-keto carboxylic acid of formula:

wherein R is H, CH₃, or CH₂OH; the method comprising contacting pyruvate and

with a hydratase-aldolase and a quinone oxidoreductase in a culture comprising two or more non-naturally occurring microbial organisms to produce the 2-keto carboxylic acid; wherein the hydratase-aldolase and the quinone oxidoreductase are expressed by the two or more non-naturally occurring microbial organisms. 11. The method of Embodiment 10, wherein at least one of the hydratase-aldolase and the quinone oxidoreductase is exogenously expressed by the two or more non-naturally occurring microbial organisms. 12. The method of Embodiment 10, wherein the hydratase-aldolase is exogenously expressed by the two or more non-naturally occurring microbial organisms. 13. The method of Embodiment 10, wherein the quinone oxidoreductase is exogenously expressed by the two or more non-naturally occurring microbial organisms. 14. The method of Embodiment 10, wherein the quinone oxidoreductase is overexpressed by the two or more non-naturally occurring microbial organisms. 15. The method of Embodiment 10, wherein the hydratase-aldolase is exogenously expressed by the two or more non-naturally occurring microbial organisms and the quinone oxidoreductase is overexpressed by the two or more non-naturally occurring microbial organisms. 16. The method of any one of Embodiments 10-15, wherein

is 3-hydroxy-propanal. 17. The method of Embodiment 16, wherein the 3-hydroxy-propanal is produced by dehydration of glycerol by a glycerol dehydratase enzyme exogenously expressed by the two or more non-naturally occurring microbial organisms. 18. The method of any one of Embodiments 10-17, further comprising separating the 2-keto carboxylic acid from the two or more non-naturally occurring microbial organisms or a culture comprising the two or more non-naturally occurring microbial organisms. 19. The method of any one of Embodiments 1-18, wherein the hydratase-aldolase is an enzyme having an EC number 4.1.2.45 or EC number 4.1.2.34 or EC number 4.1.1.4. 20. The method of any one of Embodiments 1-18, wherein the hydratase-aldolase is an enzyme selected from the group of enzymes identified under GenBank, RefSeq, or Uniprot ID Nos. D7C0E5, P0A144, Q79EM8, A0A0N0AHI8, A0A0N1FRY3, M3DYR1, W7SU48, A0A286PH18, Q9X9Q6, Q9WXH7, A4XDS1, F2J6N9, A0A063BFL5, Q9ZHH6, A0A0C1K853, WP_034398482, PYK12191, WP_115478033, WP_028222253, WP_013654807, WP_059403060, WP_092508530, WP_116642627, WP_009770659, WP_107818191, WP_003292061, PYN48855, WP_122212965, WP_028217297, WP_034507049, KMK64081.1, WP_070028041.1, or KZL92449.1. 21. The method of any one of Embodiments 1-18, wherein the hydratase-aldolase is an enzyme comprising a sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:84, SEQ ID NO:85, or SEQ ID NO:86. 22. The method of any one of Embodiments 1-18, wherein the hydratase-aldolase has at least 50% identity to an enzyme comprising a sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:84, SEQ ID NO:85, or SEQ ID NO:86. 23. The method of any one of Embodiments 1-18, wherein the hydratase-aldolase has at least 70% identity to an enzyme comprising a sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:84, SEQ ID NO:85, or SEQ ID NO:86. 24. The method of any one of Embodiments 1-18, wherein the hydratase-aldolase has at least 90% identity to an enzyme comprising a sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:84, SEQ ID NO:85, or SEQ ID NO:86. 25. The method of any one of Embodiments 1-18, wherein the hydratase-aldolase is an enzyme selected from Tables 1, 5-8. 26. The method of any one of Embodiments 1-25, wherein the quinone oxidoreductase is an enzyme having an EC number 1.6.5 (e.g., EC 1.6.5.5). 27. The method of any one of Embodiments 1-25, wherein the quinone oxidoreductase is an enzyme comprising a sequence of SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, or SEQ ID NO:97. 28. The method of any one of Embodiments 1-25, wherein the quinone oxidoreductase has at least 50% identity to an enzyme comprising a sequence of SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, or SEQ ID NO:97. 29. The method of any one of Embodiments 1-25, wherein the quinone oxidoreductase has at least 70% identity to an enzyme comprising a sequence of SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, or SEQ ID NO:97. 30. The method of any one of Embodiments 1-25, wherein the quinone oxidoreductase has at least 90% identity to an enzyme comprising a sequence of SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, or SEQ ID NO:97. 31. The method of any one of Embodiments 1-30, wherein one or more of the hydratase-aldolase and quinone oxidoreductase further comprise one or more protein tags. 32. The method of Embodiment 31, wherein the protein tags are selected from polyhistidine tag, a GST tag (glutathione-S-transferase tag), a HA tag (hemagglutinin tag), a FLAG tag, a Myc tag, a maltose binding protein tag, a chitin binding protein tag, and a fluorescent tag. 33. The method of any one of Embodiments 1-32, wherein the pyruvate is produced from carbon sources selected from glycerol, glucose, xylose, arabinose, galactose, mannose, fructose, sucrose, and starch, or a combination of thereof. 34. The method of any one of Embodiments 1-11, wherein R is CH₂OH. 35. A method for producing 1,5-pentanediol, the method comprising

-   contacting pyruvate and 3-hydroxy-propanal with a hydratase-aldolase     and a quinone oxidoreductase to produce a 2-keto carboxylic acid of     formula:

wherein R is CH₂OH;

-   contacting the 2-keto carboxylic acid with a     2-keto-acid-decarboxylase to produce a 5-hydroxy-pentanal;

and

-   contacting the 5-hydroxy-pentanal with a primary alcohol     dehydrogenase to produce the 1,5-pentanediol, wherein the method is     performed in a culture comprising one or more non-naturally     occurring microbial organisms.     36. The method of Embodiment 35, wherein the hydratase-aldolase and     the quinone oxidoreductase are expressed by the one or more     non-naturally occurring microbial organisms.     37. The method of Embodiment 35, wherein at least one of the     hydratase-aldolase and the quinone oxidoreductase is exogenously     expressed by the one or more non-naturally occurring microbial     organisms.     38. The method of Embodiment 35, wherein the hydratase-aldolase is     exogenously expressed by the one or more non-naturally occurring     microbial organisms.     39. The method of Embodiment 35, wherein the quinone oxidoreductase     is exogenously expressed by the one or more non-naturally occurring     microbial organisms.     40. The method of Embodiment 35, wherein the quinone oxidoreductase     is overexpressed by the one or more non-naturally occurring     microbial organisms.     41. The method of any one of Embodiments 35-40, wherein the     2-keto-acid-decarboxylase and the primary alcohol dehydrogenase are     expressed by the one or more non-naturally occurring microbial     organisms.     42. The method of any one of Embodiments 35-40, wherein the     2-keto-acid-decarboxylase and the primary alcohol dehydrogenase are     exogenously expressed by the one or more non-naturally occurring     microbial organisms.     43. The method of any one of Embodiments 35-40, wherein one or more     of the 2-keto-acid-decarboxylase and the primary alcohol     dehydrogenase are overexpressed by the one or more non-naturally     occurring microbial organisms.     44. The method of any one of Embodiments 35-43, further comprising     separating the 1,5-pentanediol from the one or more non-naturally     occurring microbial organisms or a culture comprising the one or     more non-naturally occurring microbial organisms.     45. A method for producing 1,5-pentanediol, the method comprising -   contacting pyruvate and 3-hydroxy-propanal with a hydratase-aldolase     and a quinone oxidoreductase to produce a 2-keto carboxylic acid of     formula:

wherein R is CH₂OH;

-   contacting the 2-keto carboxylic acid with a     2-keto-acid-decarboxylase to produce a 5-hydroxy-pentanal;

and

-   contacting the 5-hydroxy-pentanal with a primary alcohol     dehydrogenase to produce the 1,5-pentanediol, wherein the method is     performed in a culture comprising two or more non-naturally     occurring microbial organisms.     46. The method of Embodiment 45, wherein the hydratase-aldolase and     the quinone oxidoreductase are expressed by the two or more     non-naturally occurring microbial organisms.     47. The method of Embodiment 45, wherein at least one of the     hydratase-aldolase and the quinone oxidoreductase is exogenously     expressed by the two or more non-naturally occurring microbial     organisms.     48. The method of Embodiment 45, wherein the hydratase-aldolase is     exogenously expressed by the two or more non-naturally occurring     microbial organisms.     49. The method of Embodiment 45, wherein the quinone oxidoreductase     is exogenously expressed by the two or more non-naturally occurring     microbial organisms.     50. The method of Embodiment 45, wherein the quinone oxidoreductase     is overexpressed by the two or more non-naturally occurring     microbial organisms.     51. The method of any one of Embodiments 45-50, wherein the     2-keto-acid-decarboxylase and the primary alcohol dehydrogenase are     expressed by the two or more non-naturally occurring microbial     organisms.     52. The method of any one of Embodiments 45-50, wherein the     2-keto-acid-decarboxylase and the primary alcohol dehydrogenase are     exogenously expressed by the two or more non-naturally occurring     microbial organisms.     53. The method of any one of Embodiments 45-50, wherein one or more     of the 2-keto-acid-decarboxylase and the primary alcohol     dehydrogenase are overexpressed by the two or more non-naturally     occurring microbial organisms.     54. The method of any one of Embodiments 45-53, further comprising     separating the 1,5-pentanediol from the two or more non-naturally     occurring microbial organisms or a culture comprising the two or     more non-naturally occurring microbial organisms.     55. The method of any one of Embodiments 35-54, wherein the     hydratase-aldolase is an enzyme having an EC number 4.1.2.45 or EC     number 4.1.2.34 or EC number 4.1.1.4.     56. The method of any one of Embodiments 35-54, wherein the     hydratase-aldolase is an enzyme selected from the group of enzymes     identified under GenBank, RefSeq, or Uniprot ID Nos. D7C0E5, P0A144,     Q79EM8, A0A0N0AHI8, A0A0N1FRY3, M3DYR1, W7SU48, A0A286PH18, Q9X9Q6,     Q9WXH7, A4XDS1, F2J6N9, A0A063BFL5, Q9ZHH6, A0A0C1K853,     WP_034398482, PYK12191, WP_115478033, WP_028222253, WP_013654807,     WP_059403060, WP_092508530, WP_116642627, WP_009770659,     WP_107818191, WP_003292061, PYN48855, WP_122212965, WP_028217297,     WP_034507049, KMK64081.1, WP_070028041.1, or KZL92449.1.     57. The method of any one of Embodiments 35-54, wherein the     hydratase-aldolase is an enzyme comprising a sequence of SEQ ID     NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID     NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID     NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ     ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20,     SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID     NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ     ID NO:30, SEQ ID NO:84, SEQ ID NO:85, or SEQ ID NO:86.     58. The method of any one of Embodiments 35-54, wherein the     hydratase-aldolase has at least 50% identity to an enzyme comprising     a sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4,     SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ     ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,     SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID     NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ     ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28,     SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:84, SEQ ID NO:85, or SEQ ID     NO:86.     59. The method of any one of Embodiments 35-54, wherein the     hydratase-aldolase has at least 70% identity to an enzyme comprising     a sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4,     SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ     ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,     SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID     NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ     ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28,     SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:84, SEQ ID NO:85, or SEQ ID     NO:86.     60. The method of any one of Embodiments 35-54, wherein the     hydratase-aldolase has at least 90% identity to an enzyme comprising     a sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4,     SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ     ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,     SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID     NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ     ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28,     SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:84, SEQ ID NO:85, or SEQ ID     NO:86.     61. The method of any one of Embodiments 35-54, wherein the     hydratase-aldolase is an enzyme selected from Tables 1, 5-8.     62. The method of any one of Embodiments 35-61, wherein the quinone     oxidoreductase is an enzyme having an EC number 1.6.5 (e.g., EC     1.6.5.5).     63. The method of any one of Embodiments 35-61, wherein the quinone     oxidoreductase is an enzyme comprising a sequence of SEQ ID NO:45,     SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID     NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:87, SEQ ID NO:88, SEQ     ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93,     SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, or SEQ ID NO:97.     64. The method of any one of Embodiments 35-61, wherein the quinone     oxidoreductase has at least 50% identity to an enzyme comprising a     sequence of SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48,     SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID     NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ     ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, or     SEQ ID NO:97.     65. The method of any one of Embodiments 35-61, wherein the quinone     oxidoreductase has at least 70% identity to an enzyme comprising a     sequence of SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48,     SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID     NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ     ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, or     SEQ ID NO:97.     66. The method of any one of Embodiments 35-61, wherein the quinone     oxidoreductase has at least 90% identity to an enzyme comprising a     sequence of SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48,     SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID     NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ     ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, or     SEQ ID NO:97.     67. The method of any one of Embodiments 35-66, wherein the     2-keto-acid-decarboxylase is an enzyme having an EC number 4.1.1.1;     EC number 4.1.1.2; EC number 4.1.1.3; EC number 4.1.1.4; EC number     4.1.1.5; EC number 4.1.1.6; EC number 4.1.1.7; EC number 4.1.1.11;     EC number 4.1.1.12; EC number 4.1.1.15; EC number 4.1.1.16; EC     number 4.1.1.17; EC number 4.1.1.18; EC number 4.1.1.19; EC number     4.1.1.20; EC number 4.1.1.34; EC number 4.1.1.35; EC number     4.1.1.40; EC number 4.1.1.54; EC number 4.1.1.56; EC number     4.1.1.71; EC number 4.1.1.72; EC number 4.1.1.73; EC number     4.1.1.74; EC number 4.1.1.75; or EC number 4.1.1.77.     68. The method of any one of Embodiments 35-66, wherein the     2-keto-acid-decarboxylase is an enzyme selected from the group of     enzymes identified under Uniprot ID Nos. Q6QBS4, A7M7D6, or P20906.     69. The method of any one of Embodiments 35-66, wherein the     2-keto-acid-decarboxylase has at least 50% identity to an enzyme     selected from the group of enzymes identified under Uniprot ID Nos.     Q6QBS4, A7M7D6, or P20906.     70. The method of any one of Embodiments 35-66, wherein the     2-keto-acid-decarboxylase has at least 70% identity to an enzyme     selected from the group of enzymes identified under Uniprot ID Nos.     Q6QBS4, A7M7D6, or P20906.     71. The method of any one of Embodiments 35-66, wherein the     2-keto-acid-decarboxylase has at least 90% identity to an enzyme     selected from the group of enzymes identified under Uniprot ID Nos.     Q6QBS4, A7M7D6, or P20906.     72. The method of any one of Embodiments 35-71, wherein the primary     alcohol dehydrogenase is an enzyme having an EC number 1.1.1.61.     73. The method of any one of Embodiments 35-71, wherein the primary     alcohol dehydrogenase is an enzyme selected from the group of     enzymes identified under Uniprot or GenBank ID Nos. NP_417279.1,     NP_349892.1, NP_349891.1, BAB12273.1, L21902.1, Q94B07, AAB03015.1,     NP_014032.1, NP 013892.1, NP_015019.1, NP_010996.2, ABX39192.1,     XP_001210625.1, ABO67118, ABO68223, BAE77068.1, or CAA47743.1.     74. The method of any one of Embodiments 35-71, wherein the primary     alcohol dehydrogenase is an enzyme comprising a sequence of SEQ ID     NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, or SEQ ID NO:74.     75. The method of any one of Embodiments 35-71, wherein the primary     alcohol dehydrogenase has at least 50% identity to an enzyme     comprising a sequence of SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72,     SEQ ID NO:73, or SEQ ID NO:74.     76. The method of any one of Embodiments 35-71, wherein the primary     alcohol dehydrogenase has at least 70% identity to an enzyme     comprising a sequence of SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72,     SEQ ID NO:73, or SEQ ID NO:74.     77. The method of any one of Embodiments 35-71, wherein the primary     alcohol dehydrogenase has at least 90% identity to an enzyme     comprising a sequence of SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72,     SEQ ID NO:73, or SEQ ID NO:74.     78. The method of any one of Embodiments 35-54, wherein     the hydratase-aldolase is an enzyme comprising a sequence of SEQ ID     NO:8;     the quinone oxidoreductase is an enzyme comprising a sequence of SEQ     ID NO:45;     the 2-keto-acid-decarboxylase is an enzyme comprising a sequence of     SEQ ID NO:83; and     the primary alcohol dehydrogenase is an enzyme comprising a sequence     of SEQ ID NO:70.     79. The method of any one of Embodiments 35-78, wherein one or more     of the hydratase-aldolase, quinone oxidoreductase,     2-keto-acid-decarboxylase, and primary alcohol dehydrogenase further     comprise one or more protein tags.     80. The method of Embodiment 79, wherein the protein tags are     selected from polyhistidine tag, a GST tag     (glutathione-S-transferase tag), a HA tag (hemagglutinin tag), a     FLAG tag, a Myc tag, a maltose binding protein tag, a chitin binding     protein tag, and a fluorescent tag.     81. The method of any one of Embodiments 35-80, wherein the pyruvate     is produced from carbon sources selected from glycerol, glucose,     xylose, arabinose, galactose, mannose, fructose, sucrose, and     starch, or a combination thereof 82. The method of any one of     Embodiments 35-81, wherein the 3-hydroxy-propanal is produced by     dehydration of glycerol by a glycerol dehydratase enzyme exogenously     expressed by the one or more non-naturally occurring microbial     organisms.     83. A method for producing 1,6-hexanediol, the method comprising -   contacting pyruvate and 3-hydroxy-propanal with a hydratase-aldolase     and a quinone oxidoreductase to produce a 2-keto carboxylic acid of     formula:

wherein R is CH₂OH;

-   contacting the 2-keto carboxylic acid with a     6-hydroxy-2-oxohexanoate-2-reductase to produce     2,6-dihydroxy-hexanoate; -   contacting the 2,6-dihydroxy-hexanoate with a     2,6-dihydroxy-hexanoate CoA-transferase to produce     2,6-dihydroxy-hexanoyl-CoA; -   contacting the 2,6-dihydroxy-hexanoyl-CoA with a the     2,6-dihydroxy-hexanoyl-CoA 2-dehydratase to produce     6-hydroxy-2,3-dehydro-hexanoyl-CoA; -   contacting the 6-hydroxy-2,3-dehydro-hexanoyl-CoA with a     2,3-dehydro-hexanoyl-CoA 2,3-reductase to produce     6-hydroxy-hexanoyl-CoA; -   contacting the 6-hydroxy-hexanoyl-CoA with a 6-hydroxyhexanoyl-CoA     transferase to produce 6-hydroxy-hexanoate; -   contacting the 6-hydroxy-hexanoate with a 6-hydroxyhexanoate     1-reductase to produce 6-hydroxy-hexanal; and -   contacting the 6-hydroxy-hexanal with a 6-hydroxyhexanal 1-reductase     to produce the 1,6-hexanediol, wherein the method is performed in a     culture comprising one or more non-naturally occurring microbial     organisms.     84. The method of Embodiment 83, wherein the hydratase-aldolase and     the quinone oxidoreductase are expressed by the one or more     non-naturally occurring microbial organisms.     85. The method of Embodiment 83, wherein at least one of the     hydratase-aldolase and the quinone oxidoreductase is exogenously     expressed by the one or more non-naturally occurring microbial     organisms.     86. The method of Embodiment 83, wherein the hydratase-aldolase is     exogenously expressed by the one or more non-naturally occurring     microbial organisms.     87. The method of Embodiment 83, wherein the quinone oxidoreductase     is exogenously expressed by the one or more non-naturally occurring     microbial organisms.     88. The method of Embodiment 83, wherein the quinone oxidoreductase     is overexpressed by the one or more non-naturally occurring     microbial organisms.     89. The method of any one of Embodiments 83-88, wherein the     6-hydroxy-2-oxohexanoate-2-reductase, the 2,6-dihydroxy-hexanoate     CoA-transferase, the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase, the     2,3-dehydro-hexanoyl-CoA 2,3-reductase, the 6-hydroxyhexanoyl-CoA     transferase, the 6-hydroxyhexanoate 1-reductase, and the     6-hydroxyhexanal 1-reductase are expressed by the one or more     non-naturally occurring microbial organisms.     90. The method of any one of Embodiments 83-88, wherein the     6-hydroxy-2-oxohexanoate-2-reductase, the 2,6-dihydroxy-hexanoate     CoA-transferase, the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase, the     2,3-dehydro-hexanoyl-CoA 2,3-reductase, the 6-hydroxyhexanoyl-CoA     transferase, the 6-hydroxyhexanoate 1-reductase, and the     6-hydroxyhexanal 1-reductase are exogenously expressed by the one or     more non-naturally occurring microbial organisms.     91. The method of any one of Embodiments 83-88, wherein one or more     of the 6-hydroxy-2-oxohexanoate-2-reductase, the     2,6-dihydroxy-hexanoate CoA-transferase, the     2,6-dihydroxy-hexanoyl-CoA 2-dehydratase, the     2,3-dehydro-hexanoyl-CoA 2,3-reductase, the 6-hydroxyhexanoyl-CoA     transferase, the 6-hydroxyhexanoate 1-reductase, and the     6-hydroxyhexanal 1-reductase are overexpressed by the one or more     non-naturally occurring microbial organisms.     92. The method of any one of Embodiments 83-91, further comprising     separating the 1,6-hexanediol from the one or more non-naturally     occurring microbial organisms or a culture comprising the one or     more non-naturally occurring microbial organisms.     93. A method for producing 1,6-hexanediol, the method comprising -   contacting pyruvate and 3-hydroxy-propanal with a hydratase-aldolase     and a quinone oxidoreductase to produce a 2-keto carboxylic acid of     formula:

wherein R is CH₂OH;

-   contacting the 2-keto carboxylic acid with a     6-hydroxy-2-oxohexanoate-2-reductase to produce     2,6-dihydroxy-hexanoate; -   contacting the 2,6-dihydroxy-hexanoate with a     2,6-dihydroxy-hexanoate CoA-transferase to produce     2,6-dihydroxy-hexanoyl-CoA; -   contacting the 2,6-dihydroxy-hexanoyl-CoA with a the     2,6-dihydroxy-hexanoyl-CoA 2-dehydratase to produce     6-hydroxy-2,3-dehydro-hexanoyl-CoA; -   contacting the 6-hydroxy-2,3-dehydro-hexanoyl-CoA with a     2,3-dehydro-hexanoyl-CoA 2,3-reductase to produce     6-hydroxy-hexanoyl-CoA; -   contacting the 6-hydroxy-hexanoyl-CoA with a 6-hydroxyhexanoyl-CoA     transferase to produce 6-hydroxy-hexanoate; -   contacting the 6-hydroxy-hexanoate with a 6-hydroxyhexanoate     1-reductase to produce 6-hydroxy-hexanal; and -   contacting the 6-hydroxy-hexanal with a 6-hydroxyhexanal 1-reductase     to produce the 1,6-hexanediol, wherein the method is performed in a     culture comprising two or more non-naturally occurring microbial     organisms.     94. The method of Embodiment 93, wherein the hydratase-aldolase and     the quinone oxidoreductase are expressed by the two or more     non-naturally occurring microbial organisms.     95. The method of Embodiment 93, wherein at least one of the     hydratase-aldolase and the quinone oxidoreductase is exogenously     expressed by the two or more non-naturally occurring microbial     organisms.     96. The method of Embodiment 93, wherein the hydratase-aldolase is     exogenously expressed by the two or more non-naturally occurring     microbial organisms.     97. The method of Embodiment 93, wherein the quinone oxidoreductase     is exogenously expressed by the two or more non-naturally occurring     microbial organisms.     98. The method of Embodiment 93, wherein the quinone oxidoreductase     is overexpressed by the two or more non-naturally occurring     microbial organisms.     99. The method of any one of Embodiments 93-98, wherein the     6-hydroxy-2-oxohexanoate-2-reductase, the 2,6-dihydroxy-hexanoate     CoA-transferase, the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase, the     2,3-dehydro-hexanoyl-CoA 2,3-reductase, the 6-hydroxyhexanoyl-CoA     transferase, the 6-hydroxyhexanoate 1-reductase, and the     6-hydroxyhexanal 1-reductase are expressed by the two or more     non-naturally occurring microbial organisms.     100. The method of any one of Embodiments 93-98, wherein the     6-hydroxy-2-oxohexanoate-2-reductase, the 2,6-dihydroxy-hexanoate     CoA-transferase, the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase, the     2,3-dehydro-hexanoyl-CoA 2,3-reductase, the 6-hydroxyhexanoyl-CoA     transferase, the 6-hydroxyhexanoate 1-reductase, and the     6-hydroxyhexanal 1-reductase are exogenously expressed by the two or     more non-naturally occurring microbial organisms.     101. The method of any one of Embodiments 93-98, wherein one or more     of the 6-hydroxy-2-oxohexanoate-2-reductase, the     2,6-dihydroxy-hexanoate CoA-transferase, the     2,6-dihydroxy-hexanoyl-CoA 2-dehydratase, the     2,3-dehydro-hexanoyl-CoA 2,3-reductase, the 6-hydroxyhexanoyl-CoA     transferase, the 6-hydroxyhexanoate 1-reductase, and the     6-hydroxyhexanal 1-reductase are overexpressed by the two or more     non-naturally occurring microbial organisms.     102. The method of any one of Embodiments 93-101, further comprising     separating the 1,6-hexanediol from the two or more non-naturally     occurring microbial organisms or a culture comprising the two or     more non-naturally occurring microbial organisms.     103. The method of any one of Embodiments 83-102, wherein the     hydratase-aldolase is an enzyme having an EC number 4.1.2.45 or EC     number 4.1.2.34 or EC number 4.1.1.4.     104. The method of any one of Embodiments 83-102, wherein the     hydratase-aldolase is an enzyme selected from the group of enzymes     identified under GenBank, RefSeq, or Uniprot ID Nos. D7C0E5, P0A144,     Q79EM8, A0A0N0AHI8, A0A0N1FRY3, M3DYR1, W7SU48, A0A286PH18, Q9X9Q6,     Q9WXH7, A4XDS1, F2J6N9, A0A063BFL5, Q9ZHH6, A0A0C1K853,     WP_034398482, PYK12191, WP_115478033, WP_028222253, WP_013654807,     WP_059403060, WP_092508530, WP_116642627, WP_009770659,     WP_107818191, WP_003292061, PYN48855, WP_122212965, WP_028217297,     WP_034507049, KMK64081.1, WP_070028041.1, or KZL92449.1.     105. The method of any one of Embodiments 83-102, wherein the     hydratase-aldolase is an enzyme comprising a sequence of SEQ ID     NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID     NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID     NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ     ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20,     SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID     NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ     ID NO:30, SEQ ID NO:84, SEQ ID NO:85, or SEQ ID NO:86.     106. The method of any one of Embodiments 83-102, wherein the     hydratase-aldolase has at least 50% identity to an enzyme comprising     a sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4,     SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ     ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,     SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID     NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ     ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28,     SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:84, SEQ ID NO:85, or SEQ ID     NO:86.     107. The method of any one of Embodiments 83-102, wherein the     hydratase-aldolase has at least 70% identity to an enzyme comprising     a sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4,     SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ     ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,     SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID     NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ     ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28,     SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:84, SEQ ID NO:85, or SEQ ID     NO:86.     108. The method of any one of Embodiments 83-102, wherein the     hydratase-aldolase has at least 90% identity to an enzyme comprising     a sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4,     SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ     ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,     SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID     NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ     ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28,     SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:84, SEQ ID NO:85, or SEQ ID     NO:86.     109. The method of any one of Embodiments 83-102, wherein the     hydratase-aldolase is an enzyme selected from Tables 1 and 5-8.     110. The method of any one of Embodiments 83-109, wherein the     quinone oxidoreductase is an enzyme having an EC number 1.6.5 (e.g.,     EC 1.6.5.5).     111. The method of any one of Embodiments 83-109, wherein the     quinone oxidoreductase is an enzyme comprising a sequence of SEQ ID     NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ     ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:87, SEQ ID NO:88,     SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID     NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, or SEQ ID NO:97.     112. The method of any one of Embodiments 83-109, wherein the     quinone oxidoreductase has at least 50% identity to an enzyme     comprising a sequence of SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47,     SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID     NO:52, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ     ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95,     SEQ ID NO:96, or SEQ ID NO:97.     113. The method of any one of Embodiments 83-109, wherein the     quinone oxidoreductase has at least 70% identity to an enzyme     comprising a sequence of SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47,     SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID     NO:52, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ     ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95,     SEQ ID NO:96, or SEQ ID NO:97.     114. The method of any one of Embodiments 83-109, wherein the     quinone oxidoreductase has at least 90% identity to an enzyme     comprising a sequence of SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47,     SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID     NO:52, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ     ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95,     SEQ ID NO:96, or SEQ ID NO:97.     115. The method of any one of Embodiments 83-102, wherein -   the 6-hydroxy-2-oxohexanoate-2-reductase is an enzyme having an EC     number 1.1.99.6, EC number 1.1.1.169, EC number 1.1.1.215, EC number     1.1.1.28, or EC number 1.1.1.110; -   the 2,6-dihydroxy-hexanoate CoA-transferase is an enzyme having an     EC number 2.8.3, EC number 2.8.3.1, or EC number 2.8.3.12; -   the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase is an enzyme having an     EC number 4.2.1.167; -   the 2,3-dehydro-hexanoyl-CoA 2,3-reductase is an enzyme having an EC     number 1.3.1.44; -   the 6-hydroxyhexanoyl-CoA transferase is an enzyme having an EC     number 2.8.3, EC number 2.8.3.1, or EC number 2.8.3.12; -   the 6-hydroxyhexanoate 1-reductase is an enzyme having an EC number     1.2.99.6; and -   the 6-hydroxyhexanal 1-reductase is an enzyme having an EC number     1.1.1.     116. The method of any one of Embodiments 83-102, wherein -   the 6-hydroxy-2-oxohexanoate-2-reductase is an enzyme comprising a     sequence of SEQ ID NO:53, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100,     SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, or SEQ     ID NO:105; -   the 2,6-dihydroxy-hexanoate CoA-transferase is an enzyme comprising     a sequence of SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, or SEQ ID     NO:58; -   the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase is an enzyme comprising     a sequence of SEQ ID NO:59, SEQ ID NO:61, and SEQ ID NO:63; or SEQ     ID NO:60, SEQ ID NO:62, and SEQ ID NO:64; -   the 2,3-dehydro-hexanoyl-CoA 2,3-reductase is an enzyme comprising a     sequence of SEQ ID NO:65; -   the 6-hydroxyhexanoyl-CoA transferase is an enzyme comprising a     sequence of SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, or SEQ ID     NO:58; -   the 6-hydroxyhexanoate 1-reductase is an enzyme comprising a     sequence of SEQ ID NO:66, SEQ ID NO:67, or SEQ ID NO:68; and -   the 6-hydroxyhexanal 1-reductase is an enzyme comprising a sequence     of SEQ ID NO:70.     117. The method of any one of Embodiments 83-102, wherein -   the 6-hydroxy-2-oxohexanoate-2-reductase has at least 50% identity     to an enzyme comprising a sequence of SEQ ID NO:53, SEQ ID NO:98,     SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID     NO:103, SEQ ID NO:104, or SEQ ID NO:105; -   the 2,6-dihydroxy-hexanoate CoA-transferase has at least 50%     identity to an enzyme comprising a sequence of SEQ ID NO:55, SEQ ID     NO:56, SEQ ID NO:57, or SEQ ID NO:58; -   the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase has at least 50%     identity to an enzyme comprising a sequence of SEQ ID NO:59, SEQ ID     NO:61, and SEQ ID NO:63; or SEQ ID NO:60, SEQ ID NO:62, and SEQ ID     NO:64; -   the 2,3-dehydro-hexanoyl-CoA 2,3-reductase has at least 50% identity     to an enzyme comprising a sequence of SEQ ID NO:65; -   the 6-hydroxyhexanoyl-CoA transferase has at least 50% identity to     an enzyme comprising a sequence of SEQ ID NO:55, SEQ ID NO:56, SEQ     ID NO:57, or SEQ ID NO:58; -   the 6-hydroxyhexanoate 1-reductase has at least 50% identity to an     enzyme comprising a sequence of SEQ ID NO:66, SEQ ID NO:67, or SEQ     ID NO:68; and -   the 6-hydroxyhexanal 1-reductase has at least 50% identity to an     enzyme comprising a sequence of SEQ ID NO:70.     118. The method of any one of Embodiments 83-102, wherein -   the 6-hydroxy-2-oxohexanoate-2-reductase has at least 70% identity     to an enzyme comprising a sequence of SEQ ID NO:53, SEQ ID NO:98,     SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID     NO:103, SEQ ID NO:104, or SEQ ID NO:105; -   the 2,6-dihydroxy-hexanoate CoA-transferase has at least 70%     identity to an enzyme comprising a sequence of SEQ ID NO:55, SEQ ID     NO:56, SEQ ID NO:57, or SEQ ID NO:58; -   the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase has at least 70%     identity to an enzyme comprising a sequence of SEQ ID NO:59, SEQ ID     NO:61, and SEQ ID NO:63; or SEQ ID NO:60, SEQ ID NO:62, and SEQ ID     NO:64; -   the 2,3-dehydro-hexanoyl-CoA 2,3-reductase has at least 70% identity     to an enzyme comprising a sequence of SEQ ID NO:65; -   the 6-hydroxyhexanoyl-CoA transferase has at least 70% identity to     an enzyme comprising a sequence of SEQ ID NO:55, SEQ ID NO:56, SEQ     ID NO:57, or SEQ ID NO:58; -   the 6-hydroxyhexanoate 1-reductase has at least 70% identity to an     enzyme comprising a sequence of SEQ ID NO:66, SEQ ID NO:67, or SEQ     ID NO:68; and -   the 6-hydroxyhexanal 1-reductase has at least 70% identity to an     enzyme comprising a sequence of SEQ ID NO:70.     119. The method of any one of Embodiments 83-102, wherein -   the 6-hydroxy-2-oxohexanoate-2-reductase has at least 90% identity     to an enzyme comprising a sequence of SEQ ID NO:53, SEQ ID NO:98,     SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID     NO:103, SEQ ID NO:104, or SEQ ID NO:105; -   the 2,6-dihydroxy-hexanoate CoA-transferase has at least 90%     identity to an enzyme comprising a sequence of SEQ ID NO:55, SEQ ID     NO:56, SEQ ID NO:57, or SEQ ID NO:58; -   the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase has at least 90%     identity to an enzyme comprising a sequence of SEQ ID NO:59, SEQ ID     NO:61, and SEQ ID NO:63; or SEQ ID NO:60, SEQ ID NO:62, and SEQ ID     NO:64; -   the 2,3-dehydro-hexanoyl-CoA 2,3-reductase has at least 90% identity     to an comprising a sequence of SEQ ID NO:65; -   the 6-hydroxyhexanoyl-CoA transferase has at least 90% identity to     an enzyme comprising a sequence of SEQ ID NO:55, SEQ ID NO:56, SEQ     ID NO:57, or SEQ ID NO:58; -   the 6-hydroxyhexanoate 1-reductase has at least 90% identity to an     enzyme comprising a sequence of SEQ ID NO:66, SEQ ID NO:67, or SEQ     ID NO:68; and -   the 6-hydroxyhexanal 1-reductase has at least 90% identity to an     enzyme comprising a sequence of SEQ ID NO:70.     120. The method of any one of Embodiments 83-119, wherein one or     more of the 6-hydroxy-2-oxohexanoate-2-reductase, the     2,6-dihydroxy-hexanoate CoA-transferase, the     2,6-dihydroxy-hexanoyl-CoA 2-dehydratase, the     2,3-dehydro-hexanoyl-CoA 2,3-reductase, the 6-hydroxyhexanoyl-CoA     transferase, the 6-hydroxyhexanoate 1-reductase, and the     6-hydroxyhexanal 1-reductase further comprise one or more protein     tags.     121. The method of Embodiment 120, wherein the protein tags are     selected from polyhistidine tag, a GST tag     (glutathione-S-transferase tag), a HA tag (hemagglutinin tag), a     FLAG tag, a Myc tag, a maltose binding protein tag, a chitin binding     protein tag, and a fluorescent tag.     122. The method of any one of Embodiments 83-121, wherein the     pyruvate is produced from carbon sources is selected from glycerol,     glucose, xylose, arabinose, galactose, mannose, fructose, sucrose,     and starch, or a combination thereof.     123. The method of any one of Embodiments 83-122, wherein the     3-hydroxy-propanal is produced by dehydration of glycerol by a     glycerol dehydratase enzyme exogenously expressed by the one or more     non-naturally occurring microbial organisms.     124. A method for producing 6-hydroxy-hexanoate, the method     comprising -   contacting pyruvate and 3-hydroxy-propanal with a hydratase-aldolase     and a quinone oxidoreductase to produce a 2-keto carboxylic acid of     formula:

wherein R is CH₂OH;

-   contacting the 2-keto carboxylic acid with a     6-hydroxy-2-oxohexanoate-2-reductase to produce     2,6-dihydroxy-hexanoate; -   contacting the 2,6-dihydroxy-hexanoate with a     2,6-dihydroxy-hexanoate CoA-transferase to produce     2,6-dihydroxy-hexanoyl-CoA; -   contacting the 2,6-dihydroxy-hexanoyl-CoA with a the     2,6-dihydroxy-hexanoyl-CoA 2-dehydratase to produce     6-hydroxy-2,3-dehydro-hexanoyl-CoA; -   contacting the 6-hydroxy-2,3-dehydro-hexanoyl-CoA with a     2,3-dehydro-hexanoyl-CoA 2,3-reductase to produce     6-hydroxy-hexanoyl-CoA; and -   contacting the 6-hydroxy-hexanoyl-CoA with a 6-hydroxyhexanoyl-CoA     transferase to produce the 6-hydroxy-hexanoate;     wherein the method is performed in a culture comprising one or more     non-naturally occurring microbial organisms.     125. The method of Embodiment 124, wherein the hydratase-aldolase     and the quinone oxidoreductase are expressed by the one or more     non-naturally occurring microbial organisms.     126. The method of Embodiment 124, wherein at least one of the     hydratase-aldolase and the quinone oxidoreductase is exogenously     expressed by the one or more non-naturally occurring microbial     organisms.     127. The method of Embodiment 124, wherein the hydratase-aldolase is     exogenously expressed by the one or more non-naturally occurring     microbial organisms.     128. The method of Embodiment 124, wherein the quinone     oxidoreductase is exogenously expressed by the one or more     non-naturally occurring microbial organisms.     129. The method of Embodiment 124, wherein the quinone     oxidoreductase is overexpressed by the one or more non-naturally     occurring microbial organisms.     130. The method of any one of Embodiments 124-129, wherein the     6-hydroxy-2-oxohexanoate-2-reductase, the 2,6-dihydroxy-hexanoate     CoA-transferase, the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase, the     2,3-dehydro-hexanoyl-CoA 2,3-reductase, and the     6-hydroxyhexanoyl-CoA transferase are expressed by the one or more     non-naturally occurring microbial organisms.     131. The method of any one of Embodiments 124-129, wherein the     6-hydroxy-2-oxohexanoate-2-reductase, the 2,6-dihydroxy-hexanoate     CoA-transferase, the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase, the     2,3-dehydro-hexanoyl-CoA 2,3-reductase, and the     6-hydroxyhexanoyl-CoA transferase are exogenously expressed by the     one or more non-naturally occurring microbial organisms.     132. The method of any one of Embodiments 124-129, wherein one or     more of the 6-hydroxy-2-oxohexanoate-2-reductase, the     2,6-dihydroxy-hexanoate CoA-transferase, the     2,6-dihydroxy-hexanoyl-CoA 2-dehydratase, the     2,3-dehydro-hexanoyl-CoA 2,3-reductase, and the     6-hydroxyhexanoyl-CoA transferase are overexpressed by the one or     more non-naturally occurring microbial organisms.     133. The method of any one of Embodiments 124-132, further     comprising separating the 6-hydroxy-hexanoate from the one or more     non-naturally occurring microbial organisms or a culture comprising     the one or more non-naturally occurring microbial organisms.     134. A method for producing 6-hydroxy-hexanoate, the method     comprising -   contacting pyruvate and 3-hydroxy-propanal with a hydratase-aldolase     and a quinone oxidoreductase to produce a 2-keto carboxylic acid of     formula:

wherein R is CH₂OH;

-   contacting the 2-keto carboxylic acid with a     6-hydroxy-2-oxohexanoate-2-reductase to produce     2,6-dihydroxy-hexanoate; -   contacting the 2,6-dihydroxy-hexanoate with a     2,6-dihydroxy-hexanoate CoA-transferase to produce     2,6-dihydroxy-hexanoyl-CoA; -   contacting the 2,6-dihydroxy-hexanoyl-CoA with a the     2,6-dihydroxy-hexanoyl-CoA 2-dehydratase to produce     6-hydroxy-2,3-dehydro-hexanoyl-CoA; -   contacting the 6-hydroxy-2,3-dehydro-hexanoyl-CoA with a     2,3-dehydro-hexanoyl-CoA 2,3-reductase to produce     6-hydroxy-hexanoyl-CoA; and -   contacting the 6-hydroxy-hexanoyl-CoA with a 6-hydroxyhexanoyl-CoA     transferase to produce the 6-hydroxy-hexanoate; -   wherein the method is performed in a culture comprising two or more     non-naturally occurring microbial organisms.     135. The method of Embodiment 134, wherein the hydratase-aldolase     and the quinone oxidoreductase are expressed by the two or more     non-naturally occurring microbial organisms.     136. The method of Embodiment 134, wherein at least one of the     hydratase-aldolase and the quinone oxidoreductase is exogenously     expressed by the two or more non-naturally occurring microbial     organisms.     137. The method of Embodiment 134, wherein the hydratase-aldolase is     exogenously expressed by the two or more non-naturally occurring     microbial organisms.     138. The method of Embodiment 134, wherein the quinone     oxidoreductase is exogenously expressed by the two or more     non-naturally occurring microbial organisms.     139. The method of Embodiment 134, wherein the quinone     oxidoreductase is overexpressed by the two or more non-naturally     occurring microbial organisms.     140. The method of any one of Embodiments 134-139, wherein the     6-hydroxy-2-oxohexanoate-2-reductase, the 2,6-dihydroxy-hexanoate     CoA-transferase, the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase, the     2,3-dehydro-hexanoyl-CoA 2,3-reductase, and the     6-hydroxyhexanoyl-CoA transferase are expressed by the two or more     non-naturally occurring microbial organisms.     141. The method of any one of Embodiments 134-139, wherein the     6-hydroxy-2-oxohexanoate-2-reductase, the 2,6-dihydroxy-hexanoate     CoA-transferase, the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase, the     2,3-dehydro-hexanoyl-CoA 2,3-reductase, and the     6-hydroxyhexanoyl-CoA transferase are exogenously expressed by the     two or more non-naturally occurring microbial organisms.     142. The method of any one of Embodiments 134-139, wherein one or     more of the 6-hydroxy-2-oxohexanoate-2-reductase, the     2,6-dihydroxy-hexanoate CoA-transferase, the     2,6-dihydroxy-hexanoyl-CoA 2-dehydratase, the     2,3-dehydro-hexanoyl-CoA 2,3-reductase, and the     6-hydroxyhexanoyl-CoA transferase are overexpressed by the two or     more non-naturally occurring microbial organisms.     143. The method of any one of Embodiments 134-142, further     comprising separating the 6-hydroxy-hexanoate from the two or more     non-naturally occurring microbial organisms or a culture comprising     the two or more non-naturally occurring microbial organisms.     115. The method of any one of Embodiments 83-102, wherein -   the 6-hydroxy-2-oxohexanoate-2-reductase is an enzyme having an EC     number 1.1.99.6, EC number 1.1.1.169, EC number 1.1.1.215, EC number     1.1.1.28, or EC number 1.1.1.110; -   the 2,6-dihydroxy-hexanoate CoA-transferase is an enzyme having an     EC number 2.8.3, EC number 2.8.3.1, or EC number 2.8.3.12; -   the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase is an enzyme having an     EC number 4.2.1.167; -   the 2,3-dehydro-hexanoyl-CoA 2,3-reductase is an enzyme having an EC     number 1.3.1.44; -   the 6-hydroxyhexanoyl-CoA transferase is an enzyme having an EC     number 2.8.3, EC number 2.8.3.1, or EC number 2.8.3.12; -   the 6-hydroxyhexanoate 1-reductase is an enzyme having an EC number     1.2.99.6; and -   the 6-hydroxyhexanal 1-reductase is an enzyme having an EC number     1.1.1.     116. The method of any one of Embodiments 83-102, wherein -   the 6-hydroxy-2-oxohexanoate-2-reductase is an enzyme comprising a     sequence of SEQ ID NO:53, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100,     SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, or SEQ     ID NO:105; -   the 2,6-dihydroxy-hexanoate CoA-transferase is an enzyme comprising     a sequence of SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, or SEQ ID     NO:58; -   the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase is an enzyme comprising     a sequence of SEQ ID NO:59, SEQ ID NO:61, and SEQ ID NO:63; or SEQ     ID NO:60, SEQ ID NO:62, and SEQ ID NO:64; -   the 2,3-dehydro-hexanoyl-CoA 2,3-reductase is an enzyme comprising a     sequence of SEQ ID NO:65; the 6-hydroxyhexanoyl-CoA transferase is     an enzyme comprising a sequence of SEQ ID NO:55, SEQ ID NO:56, SEQ     ID NO:57, or SEQ ID NO:58; -   the 6-hydroxyhexanoate 1-reductase is an enzyme comprising a     sequence of SEQ ID NO:66, SEQ ID NO:67, or SEQ ID NO:68; and -   the 6-hydroxyhexanal 1-reductase is an enzyme comprising a sequence     of SEQ ID NO:70.     117. The method of any one of Embodiments 83-102, wherein -   the 6-hydroxy-2-oxohexanoate-2-reductase has at least 50% identity     to an enzyme comprising a sequence of SEQ ID NO:53, SEQ ID NO:98,     SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID     NO:103, SEQ ID NO:104, or SEQ ID NO:105; -   the 2,6-dihydroxy-hexanoate CoA-transferase has at least 50%     identity to an enzyme comprising a sequence of SEQ ID NO:55, SEQ ID     NO:56, SEQ ID NO:57, or SEQ ID NO:58; -   the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase has at least 50%     identity to an enzyme comprising a sequence of SEQ ID NO:59, SEQ ID     NO:61, and SEQ ID NO:63; or SEQ ID NO:60, SEQ ID NO:62, and SEQ ID     NO:64; -   the 2,3-dehydro-hexanoyl-CoA 2,3-reductase has at least 50% identity     to an enzyme comprising a sequence of SEQ ID NO:65; -   the 6-hydroxyhexanoyl-CoA transferase has at least 50% identity to     an enzyme comprising a sequence of SEQ ID NO:55, SEQ ID NO:56, SEQ     ID NO:57, or SEQ ID NO:58; -   the 6-hydroxyhexanoate 1-reductase has at least 50% identity to an     enzyme comprising a sequence of SEQ ID NO:66, SEQ ID NO:67, or SEQ     ID NO:68; and -   the 6-hydroxyhexanal 1-reductase has at least 50% identity to an     enzyme comprising a sequence of SEQ ID NO:70.     118. The method of any one of Embodiments 83-102, wherein -   the 6-hydroxy-2-oxohexanoate-2-reductase has at least 70% identity     to an enzyme comprising a sequence of SEQ ID NO:53, SEQ ID NO:98,     SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID     NO:103, SEQ ID NO:104, or SEQ ID NO:105; -   the 2,6-dihydroxy-hexanoate CoA-transferase has at least 70%     identity to an enzyme comprising a sequence of SEQ ID NO:55, SEQ ID     NO:56, SEQ ID NO:57, or SEQ ID NO:58; -   the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase has at least 70%     identity to an enzyme comprising a sequence of SEQ ID NO:59, SEQ ID     NO:61, and SEQ ID NO:63; or SEQ ID NO:60, SEQ ID NO:62, and SEQ ID     NO:64; -   the 2,3-dehydro-hexanoyl-CoA 2,3-reductase has at least 70% identity     to an enzyme comprising a sequence of SEQ ID NO:65; -   the 6-hydroxyhexanoyl-CoA transferase has at least 70% identity to     an enzyme comprising a sequence of SEQ ID NO:55, SEQ ID NO:56, SEQ     ID NO:57, or SEQ ID NO:58; -   the 6-hydroxyhexanoate 1-reductase has at least 70% identity to an     enzyme comprising a sequence of SEQ ID NO:66, SEQ ID NO:67, or SEQ     ID NO:68; and -   the 6-hydroxyhexanal 1-reductase has at least 70% identity to an     enzyme comprising a sequence of SEQ ID NO:70.     119. The method of any one of Embodiments 83-102, wherein -   the 6-hydroxy-2-oxohexanoate-2-reductase has at least 90% identity     to an enzyme comprising a sequence of SEQ ID NO:53, SEQ ID NO:98,     SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID     NO:103, SEQ ID NO:104, or SEQ ID NO:105; -   the 2,6-dihydroxy-hexanoate CoA-transferase has at least 90%     identity to an enzyme comprising a sequence of SEQ ID NO:55, SEQ ID     NO:56, SEQ ID NO:57, or SEQ ID NO:58; -   the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase has at least 90%     identity to an enzyme comprising a sequence of SEQ ID NO:59, SEQ ID     NO:61, and SEQ ID NO:63; or SEQ ID NO:60, SEQ ID NO:62, and SEQ ID     NO:64; -   the 2,3-dehydro-hexanoyl-CoA 2,3-reductase has at least 90% identity     to an comprising a sequence of SEQ ID NO:65; -   the 6-hydroxyhexanoyl-CoA transferase has at least 90% identity to     an enzyme comprising a sequence of SEQ ID NO:55, SEQ ID NO:56, SEQ     ID NO:57, or SEQ ID NO:58; -   the 6-hydroxyhexanoate 1-reductase has at least 90% identity to an     enzyme comprising a sequence of SEQ ID NO:66, SEQ ID NO:67, or SEQ     ID NO:68; and -   the 6-hydroxyhexanal 1-reductase has at least 90% identity to an     enzyme comprising a sequence of SEQ ID NO:70.     156. The method of any one of Embodiments 124-143, wherein -   the 6-hydroxy-2-oxohexanoate-2-reductase is an enzyme having an EC     number 1.1.99.6, EC number 1.1.1.169, EC number 1.1.1.215, EC number     1.1.1.28, or EC number 1.1.1.110; -   the 2,6-dihydroxy-hexanoate CoA-transferase is an enzyme having an     EC number 2.8.3, EC number 2.8.3.1, or EC number 2.8.3.12; -   the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase is an enzyme having an     EC number 4.2.1.167; -   the 2,3-dehydro-hexanoyl-CoA 2,3-reductase is an enzyme having an EC     number 1.3.1.44; and -   the 6-hydroxyhexanoyl-CoA transferase is an enzyme having an EC     number 2.8.3, EC number 2.8.3.1, or EC number 2.8.3.12.     157. The method of any one of Embodiments 124-143, wherein -   the 6-hydroxy-2-oxohexanoate-2-reductase is an enzyme comprising a     sequence of SEQ ID NO:53, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100,     SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, or SEQ     ID NO:105; -   the 2,6-dihydroxy-hexanoate CoA-transferase is an enzyme comprising     a sequence of SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, or SEQ ID     NO:58; -   the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase is an enzyme comprising     a sequence of SEQ ID NO:59, SEQ ID NO:61, and SEQ ID NO:63; or SEQ     ID NO:60, SEQ ID NO:62, and SEQ ID NO:64; -   the 2,3-dehydro-hexanoyl-CoA 2,3-reductase is an enzyme comprising a     sequence of SEQ ID NO:65; and -   the 6-hydroxyhexanoyl-CoA transferase is an enzyme comprising a     sequence of SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, or SEQ ID     NO:58.     158. The method of any one of Embodiments 124-143, wherein -   the 6-hydroxy-2-oxohexanoate-2-reductase has at least 50% identity     to an enzyme comprising a sequence of SEQ ID NO:53, SEQ ID NO:98,     SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID     NO:103, SEQ ID NO:104, or SEQ ID NO:105; -   the 2,6-dihydroxy-hexanoate CoA-transferase has at least 50%     identity to an enzyme comprising a sequence of SEQ ID NO:55, SEQ ID     NO:56, SEQ ID NO:57, or SEQ ID NO:58; -   the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase has at least 50%     identity to an enzyme comprising a sequence of SEQ ID NO:59, SEQ ID     NO:61, and SEQ ID NO:63; or SEQ ID NO:60, SEQ ID NO:62, and SEQ ID     NO:64; -   the 2,3-dehydro-hexanoyl-CoA 2,3-reductase has at least 50% identity     to an enzyme comprising a sequence of SEQ ID NO:65; and -   the 6-hydroxyhexanoyl-CoA transferase has at least 50% identity to     an enzyme comprising a sequence of SEQ ID NO:55, SEQ ID NO:56, SEQ     ID NO:57, or SEQ ID NO:58.     159. The method of any one of Embodiments 124-143, wherein -   the 6-hydroxy-2-oxohexanoate-2-reductase has at least 70% identity     to an enzyme comprising a sequence of SEQ ID NO:53, SEQ ID NO:98,     SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID     NO:103, SEQ ID NO:104, or SEQ ID NO:105; -   the 2,6-dihydroxy-hexanoate CoA-transferase has at least 70%     identity to an enzyme comprising a sequence of SEQ ID NO:55, SEQ ID     NO:56, SEQ ID NO:57, or SEQ ID NO:58; -   the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase has at least 70%     identity to an enzyme comprising a sequence of SEQ ID NO:59, SEQ ID     NO:61, and SEQ ID NO:63; or SEQ ID NO:60, SEQ ID NO:62, and SEQ ID     NO:64; -   the 2,3-dehydro-hexanoyl-CoA 2,3-reductase has at least 70% identity     to an comprising a sequence of SEQ ID NO:65; and -   the 6-hydroxyhexanoyl-CoA transferase has at least 70% identity to     an enzyme comprising a sequence of SEQ ID NO:55, SEQ ID NO:56, SEQ     ID NO:57, or SEQ ID NO:58.     160. The method of any one of Embodiments 124-143, wherein -   the 6-hydroxy-2-oxohexanoate-2-reductase has at least 90% identity     to an enzyme comprising a sequence of SEQ ID NO:53, SEQ ID NO:98,     SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID     NO:103, SEQ ID NO:104, or SEQ ID NO:105; -   the 2,6-dihydroxy-hexanoate CoA-transferase has at least 90%     identity to an enzyme comprising a sequence of SEQ ID NO:55, SEQ ID     NO:56, SEQ ID NO:57, or SEQ ID NO:58; -   the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase has at least 90%     identity to an enzyme comprising a sequence of SEQ ID NO:59, SEQ ID     NO:61, and SEQ ID NO:63; or SEQ ID NO:60, SEQ ID NO:62, and SEQ ID     NO:64; -   the 2,3-dehydro-hexanoyl-CoA 2,3-reductase has at least 90% identity     to an enzyme comprising a sequence of SEQ ID NO:65; and -   the 6-hydroxyhexanoyl-CoA transferase has at least 90% identity to     an enzyme comprising a sequence of SEQ ID NO:55, SEQ ID NO:56, SEQ     ID NO:57, or SEQ ID NO:58.     161. The method of any one of Embodiments 124-160, wherein one or     more of the 6-hydroxy-2-oxohexanoate-2-reductase, the     2,6-dihydroxy-hexanoate CoA-transferase, the     2,6-dihydroxy-hexanoyl-CoA 2-dehydratase, the     2,3-dehydro-hexanoyl-CoA 2,3-reductase, and the     6-hydroxyhexanoyl-CoA transferase further comprise one or more     protein tags.     162. The method of Embodiment 161, wherein the protein tags are     selected from polyhistidine tag, a GST tag     (glutathione-S-transferase tag), a HA tag (hemagglutinin tag), a     FLAG tag, a Myc tag, a maltose binding protein tag, a chitin binding     protein tag, and a fluorescent tag.     163. The method of any one of Embodiments 124-162, wherein the     pyruvate is produced from carbon sources is selected from glycerol,     glucose, xylose, arabinose, galactose, mannose, fructose, sucrose,     and starch, or a combination thereof.     164. The method of any one of Embodiments 124-163, wherein the     3-hydroxy-propanal is produced by dehydration of glycerol by a     glycerol dehydratase enzyme exogenously expressed by the one or more     non-naturally occurring microbial organisms.     165. A method for producing adipic acid, the method comprising -   contacting pyruvate and 3-hydroxy-propanal with a hydratase-aldolase     and a quinone oxidoreductase to produce a 2-keto carboxylic acid of     formula:

wherein R is CH₂OH;

-   contacting the 2-keto carboxylic acid with a     6-hydroxy-2-oxohexanoate-2-reductase to produce     2,6-dihydroxy-hexanoate; -   contacting the 2,6-dihydroxy-hexanoate with a     2,6-dihydroxy-hexanoate CoA-transferase to produce     2,6-dihydroxy-hexanoyl-CoA; -   contacting the 2,6-dihydroxy-hexanoyl-CoA with a the     2,6-dihydroxy-hexanoyl-CoA 2-dehydratase to produce     6-hydroxy-2,3-dehydro-hexanoyl-CoA; -   contacting the 6-hydroxy-2,3-dehydro-hexanoyl-CoA with a     2,3-dehydro-hexanoyl-CoA 2,3-reductase to produce     6-hydroxy-hexanoyl-CoA; -   contacting the 6-hydroxy-hexanoyl-CoA with a 6-hydroxyhexanoyl-CoA     transferase to produce 6-hydroxy-hexanoate; -   contacting the 6-hydroxy-hexanoate with a 6-hydroxyhexanoate     dehydrogenase to produce 6-oxo-hexanoate; and -   contacting the 6-oxo-hexanoate with a 6-oxo-hexanoate oxidase to     produce the adipic acid, -   wherein the method is performed in a culture comprising one or more     non-naturally occurring microbial organisms.     166. The method of Embodiment 165, wherein the hydratase-aldolase     and the quinone oxidoreductase are expressed by the one or more     non-naturally occurring microbial organisms.     167. The method of Embodiment 165, wherein at least one of the     hydratase-aldolase and the quinone oxidoreductase is exogenously     expressed by the one or more non-naturally occurring microbial     organisms.     168. The method of Embodiment 165, wherein the hydratase-aldolase is     exogenously expressed by the one or more non-naturally occurring     microbial organisms.     169. The method of Embodiment 165, wherein the quinone     oxidoreductase is exogenously expressed by the one or more     non-naturally occurring microbial organisms.     170. The method of Embodiment 165, wherein the quinone     oxidoreductase is overexpressed by the one or more non-naturally     occurring microbial organisms.     171. The method of any one of Embodiments 165-170, wherein the     6-hydroxy-2-oxohexanoate-2-reductase, the 2,6-dihydroxy-hexanoate     CoA-transferase, the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase, the     2,3-dehydro-hexanoyl-CoA 2,3-reductase, the 6-hydroxyhexanoyl-CoA     transferase, the 6-hydroxyhexanoate dehydrogenase, and the     6-oxo-hexanoate oxidase are expressed by the one or more     non-naturally occurring microbial organisms.     172. The method of any one of Embodiments 165-170, wherein the     6-hydroxy-2-oxohexanoate-2-reductase, the 2,6-dihydroxy-hexanoate     CoA-transferase, the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase, the     2,3-dehydro-hexanoyl-CoA 2,3-reductase, the 6-hydroxyhexanoyl-CoA     transferase, the 6-hydroxyhexanoate dehydrogenase, and the     6-oxo-hexanoate oxidase are exogenously expressed by the one or more     non-naturally occurring microbial organisms.     173. The method of any one of Embodiments 165-170, wherein one or     more of 6-hydroxy-2-oxohexanoate-2-reductase, the     2,6-dihydroxy-hexanoate CoA-transferase, the     2,6-dihydroxy-hexanoyl-CoA 2-dehydratase, the     2,3-dehydro-hexanoyl-CoA 2,3-reductase, the 6-hydroxyhexanoyl-CoA     transferase, the 6-hydroxyhexanoate dehydrogenase, and the     6-oxo-hexanoate oxidase are overexpressed by the one or more     non-naturally occurring microbial organisms.     174. The method of any one of Embodiments 165-173, further     comprising separating the adipic acid from the one or more     non-naturally occurring microbial organisms or a culture comprising     the one or more non-naturally occurring microbial organisms.     175. A method for producing adipic acid, the method comprising -   contacting pyruvate and 3-hydroxy-propanal with a hydratase-aldolase     and a quinone oxidoreductase to produce a 2-keto carboxylic acid of     formula:

wherein R is CH₂OH;

-   contacting the 2-keto carboxylic acid with a     6-hydroxy-2-oxohexanoate-2-reductase to produce     2,6-dihydroxy-hexanoate; -   contacting the 2,6-dihydroxy-hexanoate with a     2,6-dihydroxy-hexanoate CoA-transferase to produce     2,6-dihydroxy-hexanoyl-CoA; -   contacting the 2,6-dihydroxy-hexanoyl-CoA with a the     2,6-dihydroxy-hexanoyl-CoA 2-dehydratase to produce     6-hydroxy-2,3-dehydro-hexanoyl-CoA; -   contacting the 6-hydroxy-2,3-dehydro-hexanoyl-CoA with a     2,3-dehydro-hexanoyl-CoA 2,3-reductase to produce     6-hydroxy-hexanoyl-CoA; -   contacting the 6-hydroxy-hexanoyl-CoA with a 6-hydroxyhexanoyl-CoA     transferase to produce 6-hydroxy-hexanoate; -   contacting the 6-hydroxy-hexanoate with a 6-hydroxyhexanoate     dehydrogenase to produce 6-oxo-hexanoate; and -   contacting the 6-oxo-hexanoate with a 6-oxo-hexanoate oxidase to     produce the adipic acid, wherein the method is performed in a     culture comprising two or more non-naturally occurring microbial     organisms.     176. The method of Embodiment 175, wherein the hydratase-aldolase     and the quinone oxidoreductase are expressed by the two or more     non-naturally occurring microbial organisms.     177. The method of Embodiment 175, wherein at least one of the     hydratase-aldolase and the quinone oxidoreductase is exogenously     expressed by the two or more non-naturally occurring microbial     organisms.     178. The method of Embodiment 175, wherein the hydratase-aldolase is     exogenously expressed by the two or more non-naturally occurring     microbial organisms.     179. The method of Embodiment 175, wherein the quinone     oxidoreductase is exogenously expressed by the two or more     non-naturally occurring microbial organisms.     180. The method of Embodiment 175, wherein the quinone     oxidoreductase is overexpressed by the two or more non-naturally     occurring microbial organisms.     181. The method of any one of Embodiments 175-180, wherein the     6-hydroxy-2-oxohexanoate-2-reductase, the 2,6-dihydroxy-hexanoate     CoA-transferase, the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase, the     2,3-dehydro-hexanoyl-CoA 2,3-reductase, the 6-hydroxyhexanoyl-CoA     transferase, the 6-hydroxyhexanoate dehydrogenase, and the     6-oxo-hexanoate oxidase are expressed by the two or more     non-naturally occurring microbial organisms.     182. The method of any one of Embodiments 175-180, wherein the     6-hydroxy-2-oxohexanoate-2-reductase, the 2,6-dihydroxy-hexanoate     CoA-transferase, the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase, the     2,3-dehydro-hexanoyl-CoA 2,3-reductase, the 6-hydroxyhexanoyl-CoA     transferase, the 6-hydroxyhexanoate dehydrogenase, and the     6-oxo-hexanoate oxidase are exogenously expressed by the two or more     non-naturally occurring microbial organisms.     183. The method of any one of Embodiments 175-180, wherein one or     more of 6-hydroxy-2-oxohexanoate-2-reductase, the     2,6-dihydroxy-hexanoate CoA-transferase, the     2,6-dihydroxy-hexanoyl-CoA 2-dehydratase, the     2,3-dehydro-hexanoyl-CoA 2,3-reductase, the 6-hydroxyhexanoyl-CoA     transferase, the 6-hydroxyhexanoate dehydrogenase, and the     6-oxo-hexanoate oxidase are overexpressed by the two or more     non-naturally occurring microbial organisms.     184. The method of any one of Embodiments 175-183, further     comprising separating the adipic acid from the two or more     non-naturally occurring microbial organisms or a culture comprising     the two or more non-naturally occurring microbial organisms.     185. The method of any one of Embodiments 165-184, wherein the     hydratase-aldolase is an enzyme having an EC number 4.1.2.45 or EC     number 4.1.2.34 or EC number 4.1.1.4.     186. The method of any one of Embodiments 165-184, wherein the     hydratase-aldolase is an enzyme selected from the group of enzymes     identified under GenBank, RefSeq, or Uniprot ID Nos. D7C0E5, P0A144,     Q79EM8, A0A0N0AHI8, A0A0N1FRY3, M3DYR1, W7SU48, A0A286PH18, Q9X9Q6,     Q9WXH7, A4XDS1, F2J6N9, A0A063BFL5, Q9ZHH6, A0A0C1K853,     WP_034398482, PYK12191, WP_115478033, WP_028222253, WP_013654807,     WP_059403060, WP_092508530, WP_116642627, WP_009770659,     WP_107818191, WP_003292061, PYN48855, WP_122212965, WP_028217297,     WP_034507049, KMK64081.1, WP_070028041.1, or KZL92449.1.     187. The method of any one of Embodiments 165-184, wherein the     hydratase-aldolase is an enzyme comprising a sequence of SEQ ID     NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID     NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID     NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ     ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20,     SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID     NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ     ID NO:30, SEQ ID NO:84, SEQ ID NO:85, or SEQ ID NO:86.     188. The method of any one of Embodiments 165-184, wherein the     hydratase-aldolase has at least 50% identity to an enzyme comprising     a sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4,     SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ     ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,     SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID     NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ     ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28,     SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:84, SEQ ID NO:85, or SEQ ID     NO:86.     189. The method of any one of Embodiments 165-184, wherein the     hydratase-aldolase has at least 70% identity to an enzyme comprising     a sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4,     SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ     ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,     SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID     NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ     ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28,     SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:84, SEQ ID NO:85, or SEQ ID     NO:86.     190. The method of any one of Embodiments 165-184, wherein the     hydratase-aldolase has at least 90% identity to an enzyme comprising     a sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4,     SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ     ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,     SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID     NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ     ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28,     SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:84, SEQ ID NO:85, or SEQ ID     NO:86.     191. The method of any one of Embodiments 165-184, wherein the     hydratase-aldolase is an enzyme selected from Tables 1 and 5-8.     192. The method of any one of Embodiments 165-191, wherein the     quinone oxidoreductase is an enzyme having an EC number 1.6.5 (e.g.,     EC 1.6.5.5).     193. The method of any one of Embodiments 165-191, wherein the     quinone oxidoreductase is an enzyme comprising a sequence of SEQ ID     NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ     ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:87, SEQ ID NO:88,     SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID     NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, or SEQ ID NO:97.     194. The method of any one of Embodiments 165-191, wherein the     quinone oxidoreductase has at least 50% identity to an enzyme     comprising a sequence of SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47,     SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID     NO:52, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ     ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95,     SEQ ID NO:96, or SEQ ID NO:97.     195. The method of any one of Embodiments 165-191, wherein the     quinone oxidoreductase has at least 70% identity to an enzyme     comprising a sequence of SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47,     SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID     NO:52, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ     ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95,     SEQ ID NO:96, or SEQ ID NO:97.     196. The method of any one of Embodiments 165-191, wherein the     quinone oxidoreductase has at least 90% identity to an enzyme     comprising a sequence of SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47,     SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID     NO:52, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ     ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95,     SEQ ID NO:96, or SEQ ID NO:97.     197. The method of any one of Embodiments 165-184, wherein -   the 6-hydroxy-2-oxohexanoate-2-reductase is an enzyme having an EC     number 1.1.99.6, EC number 1.1.1.169, EC number 1.1.1.215, EC number     1.1.1.28, or EC number 1.1.1.110; -   the 2,6-dihydroxy-hexanoate CoA-transferase is an enzyme having an     EC number 2.8.3, EC number 2.8.3.1, or EC number 2.8.3.12; -   the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase is an enzyme having an     EC number 4.2.1.167; -   the 2,3-dehydro-hexanoyl-CoA 2,3-reductase is an enzyme having an EC     number 1.3.1.44; -   the 6-hydroxyhexanoyl-CoA transferase is an enzyme having an EC     number 2.8.3, EC number 2.8.3.1, or EC number 2.8.3.12; -   the 6-hydroxyhexanoate dehydrogenase is an enzyme having an EC     number 1.1.1.258; and -   the 6-oxo-hexanoate oxidase is an enzyme having an EC number     1.2.1.63.     198. The method of any one of Embodiments 165-184, wherein -   the 6-hydroxy-2-oxohexanoate-2-reductase is an enzyme comprising a     sequence of SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:98, SEQ ID NO:99,     SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID     NO:104, or SEQ ID NO:105; -   the 2,6-dihydroxy-hexanoate CoA-transferase is an enzyme comprising     a sequence of SEQ ID NO:55 or SEQ ID NO:58; -   the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase is an enzyme comprising     a sequence of SEQ ID NO:59, SEQ ID NO:61, and SEQ ID NO:63; or SEQ     ID NO:60, SEQ ID NO:62, and SEQ ID NO:64; -   the 2,3-dehydro-hexanoyl-CoA 2,3-reductase is an enzyme comprising a     sequence of SEQ ID NO:65; the 6-hydroxyhexanoyl-CoA transferase is     an enzyme comprising a sequence of SEQ ID NO:55 or SEQ ID NO:58; -   the 6-hydroxyhexanoate dehydrogenase is an enzyme identified     comprising a sequence of SEQ ID NO:71 or SEQ ID NO:72; and -   the 6-oxo-hexanoate oxidase is an enzyme comprising a sequence of     SEQ ID NO:75.     199. The method of any one of Embodiments 165-184, wherein -   the 6-hydroxy-2-oxohexanoate-2-reductase has at least 50% identity     to an enzyme comprising a sequence of SEQ ID NO:53, SEQ ID NO:54,     SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID     NO:102, SEQ ID NO:103, SEQ ID NO:104, or SEQ ID NO:105; -   the 2,6-dihydroxy-hexanoate CoA-transferase has at least 50%     identity to an enzyme comprising a sequence of SEQ ID NO:55 or SEQ     ID NO:58; -   the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase has at least 50%     identity to an enzyme comprising a sequence of SEQ ID NO:59, SEQ ID     NO:61, and SEQ ID NO:63; or SEQ ID NO:60, SEQ ID NO:62, and SEQ ID     NO:64; -   the 2,3-dehydro-hexanoyl-CoA 2,3-reductase has at least 50% identity     to an enzyme comprising a sequence of SEQ ID NO:65; -   the 6-hydroxyhexanoyl-CoA transferase has at least 50% identity to     an enzyme comprising a sequence of SEQ ID NO:55 or SEQ ID NO:58; -   the 6-hydroxyhexanoate dehydrogenase has at least 50% identity to an     enzyme comprising a sequence of SEQ ID NO:71 or SEQ ID NO:72; and -   the 6-oxo-hexanoate oxidase has at least 50% identity to an enzyme     comprising a sequence of SEQ ID NO:75.     200. The method of any one of Embodiments 165-184, wherein -   the 6-hydroxy-2-oxohexanoate-2-reductase has at least 70% identity     to an enzyme comprising a sequence of SEQ ID NO:53, SEQ ID NO:54,     SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID     NO:102, SEQ ID NO:103, SEQ ID NO:104, or SEQ ID NO:105; -   the 2,6-dihydroxy-hexanoate CoA-transferase has at least 70%     identity to an enzyme comprising a sequence of SEQ ID NO:55 or SEQ     ID NO:58; -   the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase has at least 70%     identity to an enzyme comprising a sequence of SEQ ID NO:59, SEQ ID     NO:61, and SEQ ID NO:63; or SEQ ID NO:60, SEQ ID NO:62, and SEQ ID     NO:64; -   the 2,3-dehydro-hexanoyl-CoA 2,3-reductase has at least 70% identity     to an comprising a sequence of SEQ ID NO:65; -   the 6-hydroxyhexanoyl-CoA transferase has at least 70% identity to     an enzyme comprising a sequence of SEQ ID NO:55 or SEQ ID NO:58; -   the 6-hydroxyhexanoate dehydrogenase has at least 70% identity to an     enzyme comprising a sequence of SEQ ID NO:71 or SEQ ID NO:72; and -   the 6-oxo-hexanoate oxidase has at least 70% identity to an enzyme     comprising a sequence of SEQ ID NO:75.     201. The method of any one of Embodiments 165-184, wherein -   the 6-hydroxy-2-oxohexanoate-2-reductase has at least 90% identity     to an enzyme comprising a sequence of SEQ ID NO:53, SEQ ID NO:54,     SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID     NO:102, SEQ ID NO:103, SEQ ID NO:104, or SEQ ID NO:105; -   the 2,6-dihydroxy-hexanoate CoA-transferase has at least 90%     identity to an enzyme comprising a sequence of SEQ ID NO:55 or SEQ     ID NO:58; -   the 2,6-dihydroxy-hexanoyl-CoA 2-dehydratase has at least 90%     identity to an enzyme comprising a sequence of SEQ ID NO:59, SEQ ID     NO:61, and SEQ ID NO:63; or SEQ ID NO:60, SEQ ID NO:62, and SEQ ID     NO:64; -   the 2,3-dehydro-hexanoyl-CoA 2,3-reductase has at least 90% identity     to an comprising a sequence of SEQ ID NO:65; -   the 6-hydroxyhexanoyl-CoA transferase has at least 90% identity to     an enzyme comprising a sequence of SEQ ID NO:55 or SEQ ID NO:58; -   the 6-hydroxyhexanoate dehydrogenase has at least 90% identity to an     enzyme comprising a sequence of SEQ ID NO:71 or SEQ ID NO:72; and -   the 6-oxo-hexanoate oxidase has at least 90% identity to an enzyme     comprising a sequence of SEQ ID NO:75.     202. The method of any one of Embodiments 165-201, wherein one or     more of the 6-hydroxy-2-oxohexanoate-2-reductase, the     2,6-dihydroxy-hexanoate CoA-transferase, the     2,6-dihydroxy-hexanoyl-CoA 2-dehydratase, the     2,3-dehydro-hexanoyl-CoA 2,3-reductase, the 6-hydroxyhexanoyl-CoA     transferase, 6-hydroxyhexanoate dehydrogenase, and the     6-oxo-hexanoate oxidase are further comprise one or more protein     tags.     203. The method of Embodiment 202, wherein the protein tags are     selected from polyhistidine tag, a GST tag     (glutathione-S-transferase tag), a HA tag (hemagglutinin tag), a     FLAG tag, a Myc tag, a maltose binding protein tag, a chitin binding     protein tag, and a fluorescent tag.     204. The method of any one of Embodiments 165-203, wherein the     pyruvate is produced from carbon sources is selected from glycerol,     glucose, xylose, arabinose, galactose, mannose, fructose, sucrose,     and starch, or a combination thereof 205. The method of any one of     Embodiments 165-204, wherein the 3-hydroxy-propanal is produced by     dehydration of glycerol by a glycerol dehydratase enzyme exogenously     expressed by the one or more non-naturally occurring microbial     organisms.     206. A recombinant microbial organism comprising a first exogenous     nucleic acid encoding an aldolase hydratase enzyme, wherein the     recombinant microbial organism is further modified to express an     increased amount of quinone oxidoreductase as compared to wild-type     or the same microbial organism that is not modified, and optionally     wherein the microbial organism is Corynebacterium glutamicum, a     clostridium species, or E. coli.     207. The recombinant microorganism of Embodiment 206, wherein the     organism comprises a second exogenous nucleic acid encoding quinone     oxidoreductase.     208. The recombinant microorganism of Embodiment 207, wherein the     first and/or second exogenous nucleic acid further comprises a     regulatory element that drives expression of the second exogenous     nucleic acid.     209. The recombinant microorganism of Embodiment 208, wherein the     regulatory element is selected from a promoter or an enhancer.     210. The recombinant microbial organism of any one of Embodiments     206-209, wherein the aldolase hydratase enzyme has an EC number     4.1.2.45 or EC number 4.1.2.34 or EC number 4.1.1.4.     211. The recombinant microbial organism of any one of Embodiments     206-209, wherein the aldolase hydratase enzyme is an enzyme selected     from the group of enzymes identified under Genbank or RefSeq or     Uniprot ID Nos. D7C0E5, P0A144, Q79EM8, A0A0N0AHI8, A0A0N1FRY3,     M3DYR1, W7SU48, A0A286PH18, Q9X9Q6, Q9WXH7, A4XDS1, F2J6N9,     A0A063BFL5, Q9ZHH6, A0A0C1K853, WP_034398482, PYK12191,     WP_115478033, WP_028222253, WP_013654807, WP_059403060,     WP_092508530, WP_116642627, WP_009770659, WP_107818191,     WP_003292061, PYN48855, WP_122212965, WP_028217297, WP_034507049,     KMK64081.1, WP_070028041.1, or KZL92449.1.     212. The recombinant microbial organism of any one of Embodiments     206-209, wherein the aldolase hydratase enzyme is an enzyme     comprising a sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ     ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID     NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID     NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ     ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23,     SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID     NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:84, SEQ ID NO:85, or     SEQ ID NO:86.     213. The recombinant microbial organism of any one of Embodiments     206-209, wherein the aldolase hydratase enzyme has at least 50%     identity to an enzyme comprising a sequence of SEQ ID NO:1, SEQ ID     NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID     NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID     NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ     ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21,     SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID     NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ     ID NO:84, SEQ ID NO:85, or SEQ ID NO:86.     214. The recombinant microbial organism of any one of Embodiments     206-209, wherein the aldolase hydratase enzyme has at least 70%     identity to an enzyme comprising a sequence of SEQ ID NO:1, SEQ ID     NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID     NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID     NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ     ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21,     SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID     NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ     ID NO:84, SEQ ID NO:85, or SEQ ID NO:86.     215. The recombinant microbial organism of any one of Embodiments     206-209, wherein the aldolase hydratase enzyme has at least 90%     identity to an enzyme comprising a sequence of SEQ ID NO:1, SEQ ID     NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID     NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID     NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ     ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21,     SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID     NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ     ID NO:84, SEQ ID NO:85, or SEQ ID NO:86.     216. The recombinant microbial organism of any one of Embodiments     206-209, wherein the aldolase hydratase enzyme is an enzyme selected     from Tables 1, 5-8.     217. The recombinant microbial organism of any one of Embodiments     206-216, wherein the first exogenous nucleic acid and the second     exogenous nucleic acid are each contained in a vector.     218. The recombinant microbial organism of Embodiment 217, wherein     the first exogenous nucleic acid and the second exogenous nucleic     acid are each contained in the same vector.     219. The recombinant microbial organism of Embodiment 218, wherein     the first exogenous nucleic acid and the second exogenous nucleic     acid are each contained in their own separate vectors.     220. The recombinant microbial organism of any one of Embodiments     217-219, wherein the vector is a plasmid.     221. The recombinant microbial organism of any one of Embodiments     206-220, wherein the quinone oxidoreductase is an enzyme having an     EC number 1.6.5 (e.g., EC 1.6.5.5).     222. The recombinant microbial organism of any one of Embodiments     206-220, wherein the quinone oxidoreductase is an enzyme comprising     a sequence of SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID     NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ     ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91,     SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID     NO:96, or SEQ ID NO:97.     223. The recombinant microbial organism of any one of Embodiments     206-220, wherein the quinone oxidoreductase has at least 50%     identity to an enzyme comprising a sequence of SEQ ID NO:45, SEQ ID     NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ     ID NO:51, SEQ ID NO:52, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89,     SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID     NO:94, SEQ ID NO:95, SEQ ID NO:96, or SEQ ID NO:97.     224. The recombinant microbial organism of any one of Embodiments     206-220, wherein the quinone oxidoreductase has at least 70%     identity to an enzyme comprising a sequence of SEQ ID NO:45, SEQ ID     NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ     ID NO:51, SEQ ID NO:52, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89,     SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID     NO:94, SEQ ID NO:95, SEQ ID NO:96, or SEQ ID NO:97.     225. The recombinant microbial organism of any one of Embodiments     206-220, wherein the quinone oxidoreductase has at least 90%     identity to an enzyme comprising a sequence of SEQ ID NO:45, SEQ ID     NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ     ID NO:51, SEQ ID NO:52, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89,     SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID     NO:94, SEQ ID NO:95, SEQ ID NO:96, or SEQ ID NO:97.     226. The recombinant microbial organism of any one of Embodiments     206-220, wherein one or more of the hydratase-aldolase enzyme and     quinone oxidoreductase further comprise one or more protein tags.     227. The recombinant microbial organism of Embodiment 226, wherein     the protein tags are selected from polyhistidine tag, a GST tag     (glutathione-S-transferase tag), a HA tag (hemagglutinin tag), a     FLAG tag, a Myc tag, a maltose binding protein tag, a chitin binding     protein tag, and a fluorescent tag.     228. The recombinant microbial organism of any one of Embodiments     206-227, wherein the recombinant microbial organism is capable of     producing a 2-keto carboxylic acid of formula:

wherein R is H, CH₃, or CH₂OH. 229. The recombinant microbial organism of any one of Embodiments 206-228, wherein the recombinant microbial organism is capable of producing 1,5-pentanediol, 1,6-hexanediol, adipic acid, or 6-hydroxy hexanoate. 230. The recombinant microbial organism of any one of Embodiments 206-229, wherein the recombinant microbial organism is genetically modified to improve production of pyruvate from a carbon source. 231. The recombinant microbial organism of Embodiment 230, wherein the carbon source is selected from glycerol, glucose, xylose, arabinose, galactose, mannose, fructose, sucrose, and starch, or a combination thereof. 232. A population of recombinant microbial organisms of any one of Embodiments 206-231. 233. The population of Embodiment 232, which is substantially homogenous. 234. A method of producing 1,5-pentanediol, 1,6-hexanediol, adipic acid, or 6-hydroxy hexanoate, comprising culturing the population of Embodiment 232 or Embodiment 233 under suitable conditions. 235. The method of Embodiment 234, further comprising isolating the 1,5-pentanediol, 1,6-hexanediol, adipic acid, or 6-hydroxy hexanoate from the culture or the microbial organisms. 236. A culture comprising the recombinant microbial organisms of any one of Embodiments 206-231. 237. A culture comprising the populations of Embodiment 232 or Embodiment 233. 238. A method comprising:

contacting pyruvate and an aldehyde with an aldol product biosynthesis polypeptide so that an aldol product is produced, wherein:

the aldol product is a compound comprising an aldehyde or ketone group and a hydroxyl group attached to a beta-carbon of an aldehyde or ketone carbonyl group.

239. The method of Embodiment 238, wherein a —CHO group of the aldehyde is not conjugated to a double bond, a triple bond or an aromatic group. 240. A method comprising:

contacting pyruvate and an aliphatic aldehyde with an aldol product biosynthesis polypeptide so that an aldol product is produced, wherein:

the carbonyl group of the aliphatic aldehyde is not conjugated to a alkenyl, alkynyl, or aromatic group; and

the aldol product is a compound comprising an aldehyde or ketone group and a hydroxyl group attached to a beta-carbon of an aldehyde or ketone carbonyl group.

241. The method of any one of Embodiments 238-240, wherein the aldol product biosynthesis polypeptide is or comprises an aldolase. 242. The method of any one of Embodiments 238-241, wherein the aldol product biosynthesis polypeptide is in a microbe. 243. The method of Embodiment 242, wherein the microbe is engineered to contain an exogenous nucleic acid that encodes an aldol product biosynthesis polypeptide. 244. The method of Embodiment any one of Embodiments 242-243, wherein the microbe expresses a modulated level of an aldol product biosynthesis polypeptide. 245. The method of Embodiment any one of Embodiments 242-244, wherein the microbe expresses an engineered aldol product biosynthesis polypeptide. 246. The method of any one of Embodiments 238-245, wherein conversion of pyruvate and an aliphatic aldehyde into an aldol product is catalyzed by an aldol product biosynthesis polypeptide. 247. The method of any one of Embodiments 238-246, wherein the method is performed in a culture. 248. The method of any one of Embodiments 238-247, comprising converting an aldol product into an aldol-dehydration product, wherein the aldol-dehydration product is a compound comprising an aldehyde or ketone group and a double bond conjugated with the aldehyde or ketone group. 249. The method of Embodiment 248, wherein the converting comprises contacting an aldol product with a dehydration product biosynthesis polypeptide so that an aldol-dehydration product is produced. 250. The method of any one of Embodiments 248-249, wherein the dehydration product biosynthesis polypeptide is in a microbe. 251. The method of Embodiment 250, wherein the microbe is engineered to contain an exogenous nucleic acid that encodes a dehydration product biosynthesis polypeptide. 252. The method of Embodiment any one of Embodiments 250-251, wherein the microbe expresses a modulated level of a dehydration product biosynthesis polypeptide. 253. The method of Embodiment any one of Embodiments 250-252, wherein the microbe expresses an engineered dehydration product biosynthesis polypeptide. 254. The method of any one of Embodiments 248-253, wherein conversion of an aldol product into an aldol-dehydration product is catalyzed by a dehydration product biosynthesis polypeptide. 255. The method of any one of Embodiments 248-254, wherein the method is performed in a culture. 256. The method of Embodiment 249, wherein a dehydration product biosynthesis polypeptide is a dehydratase. 257. A method comprising:

contacting pyruvate and an aldehyde with an aldol-dehydration product biosynthesis polypeptide so that an aldol-dehydration product is produced, wherein:

the aldol-dehydration product is a compound comprising an aldehyde or ketone group and a double bond conjugated with the aldehyde or ketone group.

258. The method of Embodiment 257, wherein a —CHO group of the aldehyde is not conjugated to a double bond, a triple bond or an aromatic group. 259. A method comprising:

contacting pyruvate and an aliphatic aldehyde with an aldol-dehydration product biosynthesis polypeptide so that an aldol-dehydration product is produced, wherein:

the carbonyl group of the aliphatic aldehyde is not conjugated to a alkenyl, alkynyl, or aromatic group; and

the aldol-dehydration product is a compound comprising an aldehyde or ketone group and a double bond conjugated with the aldehyde or ketone group.

260. The method of any one of Embodiments 257-259, wherein the aldol-dehydration product biosynthesis polypeptide is or comprises a hydratase-aldolase. 261. The method of Embodiment 260, wherein contacting pyruvate and an aliphatic aldehyde with a hydratase-aldolase produces an aldol-dehydration product. 262. The method of any one of Embodiments 257-259, wherein the aldol-dehydration product biosynthesis polypeptide is or comprises an enzyme having an EC number 4.1.2.45 or EC number 4.1.2.34, or EC 4.1.1.4, or is selected from Tables 1 and 5-8. 263. The method of any one of Embodiments 257-259, wherein the aldol-dehydration product biosynthesis polypeptide is or comprises a polypeptide which shares 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 95%, 99% or more homology with an enzyme of Embodiment 262. 264. The method of any one of Embodiments 257-259, wherein the aldol-dehydration product biosynthesis polypeptide is or comprises an aldolase. 265. The method of any one of Embodiments 257-264, wherein the aldol-dehydration product biosynthesis polypeptide is in a microbe. 266. The method of Embodiment 265, wherein the microbe is engineered to contain an exogenous nucleic acid that encodes an aldol-dehydration product biosynthesis polypeptide. 267. The method of Embodiment any one of Embodiments 265-266, wherein the microbe expresses a modulated level of an aldol-dehydration product biosynthesis polypeptide. 268. The method of Embodiment any one of Embodiments 265-267, wherein the microbe expresses an engineered aldol-dehydration product biosynthesis polypeptide. 269. The method of any one of Embodiments 257-268, wherein conversion of pyruvate and an aliphatic aldehyde into an aldol-dehydration product is catalyzed by an aldol-dehydration product biosynthesis polypeptide. 270. The method of any one of Embodiments 257-269, wherein the method is performed in a culture. 271. A method comprising:

contacting an alkene with an alkene reduction product biosynthesis polypeptide so that an alkene reduction product is produced, wherein:

the alkene comprises a double bond conjugated to a carbonyl group; and

a double bond conjugated to a carbonyl group in the alkene is reduced to a single bond to provide an alkene reduction product.

272. The method of Embodiment 271, wherein the alkene is an aldol-dehydration product of any one of Embodiments 257-270. 273. The method of any one of Embodiments 271-272, wherein an alkene reduction product biosynthesis polypeptide is or comprises an enzyme that catalyzes reduction of a 2-oxo-3-enoic acid or a salt thereof. 274. The method of any one of Embodiments 271-272, wherein an alkene reduction product biosynthesis polypeptide is or comprises an enzyme that belongs to EC 1.6.5. 275. The method of any one of Embodiments 271-272, wherein an alkene reduction product biosynthesis polypeptide is or comprises an enzyme that belongs to EC 1.6.5.5 or is selected from Table 9. 276. The method of any one of Embodiments 271-272, wherein the alkene reduction product biosynthesis polypeptide is or comprises a polypeptide which shares 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 95%, 99% or more homology with an enzyme of any one of Embodiments 274-275. 277. The method of any one of Embodiments 271-276, wherein an alkene reduction product biosynthesis polypeptide is in a microbe. 278. The method of Embodiment 277, wherein the microbe is engineered to contain an exogenous nucleic acid that encodes an alkene reduction product biosynthesis polypeptide. 279. The method of Embodiment any one of Embodiments 277-278, wherein the microbe expresses a modulated level of an alkene reduction product biosynthesis polypeptide. 280. The method of Embodiment any one of Embodiments 277-279, wherein the microbe expresses an engineered alkene reduction product biosynthesis polypeptide. 281. The method of any one of Embodiments 271-280, wherein conversion of an alkene into an alkene reduction product is catalyzed by an alkene reduction product biosynthesis polypeptide. 282. The method of any one of Embodiments 271-281, wherein the method is performed in a culture. 283. The method of any one of Embodiments 238-270, comprising a method of any one of Embodiments 271-282. 284. The method of any one of Embodiments 238-283, wherein the aldehyde has the structure of formula A-1 thereof:

R^(a)-L²-O—C(O)H,   A-1

or a salt thereof, wherein:

R^(a) is R″ or —OR″,

each of L¹ and L² is independently a covalent bond, or a bivalent, optionally substituted, linear or branched C₁₋₂₀ aliphatic or C₁₋₂₀ heteroaliphatic, wherein one or more methylene units are optionally and independently replaced by —C≡C—, —C(R″)₂—, -Cy-, —O—, —S—, —S—S—, —N(R″)—, —C(O)—, —C(S)—, —C(NR″)—, —C(O)N(R″)—, —N(R″)C(O)N(R″)—, —N(R″)C(O)O—, —S(O)—, —S(O)₂—, —S(O)₂N(R″)—, —C(O)₅—, or —C(O)O—;

-Cy- is a bivalent, optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring, wherein each monocyclic ring is independently an optionally substituted, saturated, partially saturated or aromatic 3-20 membered ring having 0-5 heteroatoms;

each R″ is independently —R′, —C(O)R′, —CO₂R′, or —SO₂R′;

R′ is hydrogen, or an optionally substituted group selected from C₁₋₁₀ aliphatic, C₁₋₁₀ heteroaliphatic having 1-5 heteroatoms, a 6-10 membered aryl ring, a 5-10 membered heteroaryl ring having 1-5 heteroatoms, and a 3-10 membered heterocyclic ring having 1-5 heteroatoms, or:

two or more R′ groups are taken together with their intervening atoms to form an optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring having, in addition to the intervening atoms, 0-5 heteroatoms, wherein each monocyclic ring is independently an optionally substituted, saturated, partially saturated or aromatic 3-20 membered ring having 0-5 heteroatoms.

285. The method of any one of Embodiments 238-256 and 284, wherein the aldol product has the structure of formula P-1:

R^(a)-L²-L¹-CH(OH)—CH₂—C(O)—C(O)OH,   P-1

or a salt thereof, wherein:

R^(a) is R″ or —OR″,

each of L¹ and L² is independently a covalent bond, or a bivalent, optionally substituted, linear or branched C₁₋₂₀ aliphatic or C₁₋₂₀ heteroaliphatic, wherein one or more methylene units are optionally and independently replaced by —C≡C—, —C(R″)₂—, -Cy-, —O—, —S—, —S—S—, —N(R″)—, —C(O)—, —C(S)—, —C(NR″)—, —C(O)N(R″)—, —N(R″)C(O)N(R″)—, —N(R″)C(O)O—, —S(O)—, —S(O)₂—, —S(O)₂N(R″)—, —C(O)S—, or —C(O)O—;

-Cy- is a bivalent, optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring, wherein each monocyclic ring is independently an optionally substituted, saturated, partially saturated or aromatic 3-20 membered ring having 0-5 heteroatoms;

each R″ is independently —R′, —C(O)R′, —CO₂R′, or —SO₂R′;

R′ is hydrogen, or an optionally substituted group selected from C₁₋₁₀ aliphatic, C₁₋₁₀ heteroaliphatic having 1-5 heteroatoms, a 6-10 membered aryl ring, a 5-10 membered heteroaryl ring having 1-5 heteroatoms, and a 3-10 membered heterocyclic ring having 1-5 heteroatoms, or:

two or more R′ groups are taken together with their intervening atoms to form an optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring having, in addition to the intervening atoms, 0-5 heteroatoms, wherein each monocyclic ring is independently an optionally substituted, saturated, partially saturated or aromatic 3-20 membered ring having 0-5 heteroatoms.

286. The method of any one of Embodiments 257-285, wherein the aldol-dehydration product has the structure of formula P-2:

R^(a)-L²-O—CH═CH—C(O)—C(O)OH,   P-2

or a salt thereof, wherein:

R^(a) is R″ or —OR″,

each of L¹ and L² is independently a covalent bond, or a bivalent, optionally substituted, linear or branched C₁₋₂₀ aliphatic or C₁₋₂₀ heteroaliphatic, wherein one or more methylene units are optionally and independently replaced by —C≡C—, —C(R″)₂—, -Cy-, —O—, —S—, —S—S—, —N(R″)—, —C(O)—, —C(S)—, —C(NR″)—, —C(O)N(R″)—, —N(R″)C(O)N(R″)—, —N(R″)C(O)O—, —S(O)—, —S(O)₂—, —S(O)₂N(R″)—, —C(O)S—, or —C(O)O—;

-Cy- is a bivalent, optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring, wherein each monocyclic ring is independently an optionally substituted, saturated, partially saturated or aromatic 3-20 membered ring having 0-5 heteroatoms;

each R″ is independently —R′, —C(O)R′, —CO₂R′, or —SO₂R′;

R′ is hydrogen, or an optionally substituted group selected from C₁₋₁₀ aliphatic, C₁₋₁₀ heteroaliphatic having 1-5 heteroatoms, a 6-10 membered aryl ring, a 5-10 membered heteroaryl ring having 1-5 heteroatoms, and a 3-10 membered heterocyclic ring having 1-5 heteroatoms, or:

two or more R′ groups are taken together with their intervening atoms to form an optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring having, in addition to the intervening atoms, 0-5 heteroatoms, wherein each monocyclic ring is independently an optionally substituted, saturated, partially saturated or aromatic 3-20 membered ring having 0-5 heteroatoms.

287. The method of Embodiment 286, wherein the —CH═CH— is in E configuration. 288. The method of Embodiment 286, wherein the —CH═CH— is in Z configuration. 289. The method of any one of Embodiments 271-288, wherein the alkene reduction product has the structure of formula P-3:

R^(a)-L²-O—CH₂—CH₂—C(O)—C(O)OH,   P-3

or a salt thereof, wherein:

R^(a) is R″ or —OR″,

each of L¹ and L² is independently a covalent bond, or a bivalent, optionally substituted, linear or branched C₁₋₂₀ aliphatic or C₁₋₂₀ heteroaliphatic, wherein one or more methylene units are optionally and independently replaced by —C≡C—, —C(R″)₂—, -Cy-, —O—, —S—, —S—S—, —N(R″)—, —C(O)—, —C(S)—, —C(NR″)—, —C(O)N(R″)—, —N(R″)C(O)N(R″)—, —N(R″)C(O)O—, —S(O)—, —S(O)₂—, —S(O)₂N(R″)—, —C(O)S—, or —C(O)O—;

-Cy- is a bivalent, optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring, wherein each monocyclic ring is independently an optionally substituted, saturated, partially saturated or aromatic 3-20 membered ring having 0-5 heteroatoms;

each R″ is independently —R′, —C(O)R′, —CO₂R′, or —SO₂R′;

R′ is hydrogen, or an optionally substituted group selected from C₁₋₁₀ aliphatic, C₁₋₁₀ heteroaliphatic having 1-5 heteroatoms, a 6-10 membered aryl ring, a 5-10 membered heteroaryl ring having 1-5 heteroatoms, and a 3-10 membered heterocyclic ring having 1-5 heteroatoms, or:

two or more R′ groups are taken together with their intervening atoms to form an optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring having, in addition to the intervening atoms, 0-5 heteroatoms, wherein each monocyclic ring is independently an optionally substituted, saturated, partially saturated or aromatic 3-20 membered ring having 0-5 heteroatoms.

290. The method of any one of Embodiments 238-284, comprising converting an alkene reduction product into a compound of formula P-10:

HO—C(O)-L^(2′)-L¹-CH₂—CH₂—CH₂—C(O)—OH,   P-10

or a salt thereof. 291. The method of any one of Embodiments 238-284, comprising converting an alkene reduction product into a compound of formula P-10′:

R^(a)-L²-L¹-CH₂—CH₂—CH₂—CH₂—OH,   P-10′

or a salt thereof. 292. The method of any one of Embodiments 238-291, comprising converting an alkene reduction product into a carbonyl reduction product, wherein:

the alkene reduction product comprises a carbonyl group; and

a carbonyl group of the alkene reduction product is converted to —CH(OH)—.

293. The method of any one of Embodiments 238-291, comprising contacting an alkene reduction product with a carbonyl reduction product biosynthesis polypeptide so that a carbonyl reduction product is produced, wherein:

the alkene reduction product comprises a carbonyl group; and

a carbonyl group of the alkene reduction product is converted to —CH(OH)—.

294. The method of Embodiment 293, wherein the carbonyl reduction product biosynthesis polypeptide is or comprises a keto reductase or a 2-keto acid-2-reductase. 295. The method of any one of Embodiments 293-294, wherein the carbonyl reduction product biosynthesis polypeptide is in a microbe. 296. The method of Embodiment 295, wherein the microbe is engineered to contain an exogenous nucleic acid that encodes a carbonyl reduction product biosynthesis polypeptide. 297. The method of Embodiment any one of Embodiments 295-296, wherein the microbe expresses a modulated level of a carbonyl reduction product biosynthesis polypeptide. 298. The method of Embodiment any one of Embodiments 295-297, wherein the microbe expresses an engineered carbonyl reduction product biosynthesis polypeptide. 299. The method of any one of Embodiments 290-298, wherein conversion of an alkene reduction product into a carbonyl reduction product is catalyzed by a carbonyl reduction product biosynthesis polypeptide. 300. The method of any one of Embodiments 290-299, wherein the method is performed in a culture. 301. The method of any one of Embodiments 290-300, wherein a carbonyl reduction product has the structure of formula P-4:

R^(a)-L²-L¹-CH₂—CH₂—CH(OH)—C(O)OH,   P-4

or a salt thereof, wherein:

R^(a) is R″ or —OR″,

each of L¹ and L² is independently a covalent bond, or a bivalent, optionally substituted, linear or branched C₁₋₂₀ aliphatic or C₁₋₂₀ heteroaliphatic, wherein one or more methylene units are optionally and independently replaced by —C≡C—, —C(R″)₂—, -Cy-, —O—, —S—, —S—S—, —N(R″)—, —C(O)—, —C(S)—, —C(NR″)—, —C(O)N(R″)—, —N(R″)C(O)N(R″)—, —N(R″)C(O)O—, —S(O)—, —S(O)₂—, —S(O)₂N(R″)—, —C(O)S—, or —C(O)O—;

-Cy- is a bivalent, optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring, wherein each monocyclic ring is independently an optionally substituted, saturated, partially saturated or aromatic 3-20 membered ring having 0-5 heteroatoms;

each R″ is independently —R′, —C(O)R′, —CO₂R′, or —SO₂R′;

R′ is hydrogen, or an optionally substituted group selected from C₁₋₁₀ aliphatic, C₁₋₁₀ heteroaliphatic having 1-5 heteroatoms, a 6-10 membered aryl ring, a 5-10 membered heteroaryl ring having 1-5 heteroatoms, and a 3-10 membered heterocyclic ring having 1-5 heteroatoms, or:

two or more R′ groups are taken together with their intervening atoms to form an optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring having, in addition to the intervening atoms, 0-5 heteroatoms, wherein each monocyclic ring is independently an optionally substituted, saturated, partially saturated or aromatic 3-20 membered ring having 0-5 heteroatoms.

302. The method of any one of Embodiments 238-301, comprising converting a compound of formula P-4 or a salt thereof into a compound of formula P-5:

R^(a)-L²-L¹-CH₂—CH₂—CH(OH)—C(O)—S—CoA,   P-5

or a salt thereof. 303. The method of Embodiment 302, wherein the conversion comprises contacting a compound of formula P-4 or a salt thereof with a CoA transfer product biosynthesis polypeptide. 304. The method of any one of Embodiments 238-303, comprising converting a compound of formula P-5 or a salt thereof into a compound of formula P-6:

R^(a)-L²-L¹-CH₂—CH═CH—C(O)—S—CoA,   P-6

or a salt thereof. 305. The method of Embodiment 304, wherein the conversion comprises contacting a compound of formula P-5 or a salt thereof with a dehydration product biosynthesis polypeptide. 306. The method of any one of Embodiments 238-305, comprising converting a compound of formula P-6 or a salt thereof into a compound of formula P-7:

R^(a)-L²-L¹-CH₂—CH₂—CH₂—C(O)—S—CoA,   P-7

or a salt thereof. 307. The method of Embodiment 306, wherein the conversion comprises contacting a compound of formula P-6 or a salt thereof with a reduction product biosynthesis polypeptide which is or comprises 2,3-enoyl-CoA reductase. 308. The method of any one of Embodiments 238-307, comprising converting a compound of formula P-7 or a salt thereof into a compound of formula P-8:

R^(a)-L²-L¹-CH₂—CH₂—CH₂—C(O)—OH,   P-8

or a salt thereof. 309. The method of Embodiment 308, wherein the conversion comprises contacting a compound of formula P-7 or a salt thereof with a CoA transfer product biosynthesis polypeptide. 310. The method of any one of Embodiments 238-309, comprising converting a compound of formula P-8, wherein L² is —CH₂-L^(2′)-, or a salt thereof into a compound of formula P-9:

H—C(O)-L^(2′)-L¹-CH₂—CH₂—CH₂—C(O)—OH,   P-9

or a salt thereof, wherein:

L^(2′) is a covalent bond, or a bivalent, optionally substituted, linear or branched C₁₋₁₉ aliphatic or C₁₋₁₉ heteroaliphatic, wherein one or more methylene units are optionally and independently replaced by —C≡C—, —C(R″)₂—, -Cy-, —O—, —S—, —S—S—, —N(R″)—, —C(O)—, —C(S)—, —C(NR″)—, —C(O)N(R″)—, —N(R″)C(O)N(R″)—, —N(R″)C(O)O—, —S(O)—, —S(O)₂—, —S(O)₂N(R″)—, —C(O)S—, or —C(O)O—.

311. The method of Embodiment 310, wherein the conversion comprises contacting a compound of formula P-8 or a salt thereof with an oxidation product biosynthesis polypeptide which is or comprises an alcohol dehydrogenase. 312. The method of any one of Embodiments 238-311, comprising converting a compound of formula P-9 or a salt thereof into a compound of formula P-10:

HO—C(O)-L^(2′)-L¹-CH₂—CH₂—CH₂—C(O)—OH,   P-10

or a salt thereof. 313. The method of Embodiment 312, wherein the conversion comprises contacting a compound of formula P-9 or a salt thereof with an aldehyde oxidation product biosynthesis polypeptide. 314. The method of any one of Embodiments 238-312, comprising converting a compound of formula P-8 or a salt thereof into a compound of formula P-9′:

R^(a)-L²-L¹-CH₂—CH₂—CH₂—C(O)—H,   P-9′

or a salt thereof. 315. The method of Embodiment 314, comprising contacting a compound of formula P-8 or a salt thereof with a carboxyl reduction product biosynthesis polypeptide. 316. The method of any one of Embodiments 238-315, comprising converting a compound of formula P-9′ or a salt thereof into a compound of formula P-10′:

R^(a)-L²-L¹-CH₂—CH₂—CH₂—CH₂—OH,   P-10′

or a salt thereof. 317. The method of Embodiment 316, comprising contacting a compound of formula P-9′ or a salt thereof with an aldehyde reduction product biosynthesis polypeptide which is or comprises an aldehyde reductase or a primary alcohol dehydrogenase. 318. The method of any one of Embodiments 238-290, comprising converting a compound of formula P-3 or a salt thereof into a compound of formula P-5′:

R^(a)-L²-L¹-CH₂—CH₂—CH₂—OH,   P-5′

or a salt thereof. 319. The method of any one of Embodiments 238-290 or 318, comprising converting a compound of formula P-3 or a salt thereof into a compound of formula P-4′:

R^(a)-L²-L¹-CH₂—CH₂—C(O)—H,   P-4′

or a salt thereof. 320. The method of Embodiment 319, comprising contacting a compound of formula P-3 or a salt thereof with a decarboxylation product biosynthesis polypeptide. 321. The method of any one of Embodiments 238-290, comprising converting a compound of formula P-4′ or a salt thereof into a compound of formula P-5′:

R^(a)-L²-L¹-CH₂—CH₂—CH₂—OH,   P-5′

or a salt thereof. 322. The method of Embodiment 321, comprising contacting a compound of formula P-4′ or a salt thereof with an aldehyde reduction product biosynthesis polypeptide. 323. The method of any one of Embodiments 301-322, wherein one or more or each converting independently comprises contacting a compound with a suitable biosynthesis polypeptide. 324. The method of Embodiment 323, wherein one or more or all biosynthesis polypeptides are independently in a microbe. 325. The method of Embodiment 324, wherein the microbe is engineered to contain one or more exogenous nucleic acids that encode one or more or all of the biosynthesis polypeptides. 326. The method of Embodiment any one of Embodiments 324-325, wherein the microbe expresses a modulated level of one or more or all of the biosynthesis polypeptides. 327. The method of Embodiment any one of Embodiments 324-326, wherein one or more or all of the biosynthesis polypeptides are independently engineered. 328. The method of any one of Embodiments 324-326, wherein a suitable biosynthesis polypeptide catalyzes a corresponding conversion. 329. The method of any one of Embodiments 285-328, wherein W is —H. 330. The method of any one of Embodiments 285-328, wherein W is —OH. 331. The method of any one of Embodiments 285-330, wherein L¹ is optionally substituted C₁₋₆ alkylene. 332. The method of any one of Embodiments 285-330, wherein L¹ is unsubstituted C₁₋₆ alkylene. 333. The method of any one of Embodiments 331-332, wherein the alkylene is —CH₂—. 334. The method of any one of Embodiments 331-332, wherein the alkylene is —CH₂CH₂—. 335. The method of any one of Embodiments 331-332, wherein the alkylene is —CH₂CH₂CH₂—. 336. The method of any one of Embodiments 285-330, wherein L¹ is a covalent bond. 337. The method of any one of Embodiments 285-336, wherein L² is a covalent bond. 338. The method of any one of Embodiments 285-336, wherein L² is optionally substituted C₁₋₆ alkylene. 339. The method of any one of Embodiments 285-336, wherein L² is unsubstituted C₁₋₆ alkylene. 340. The method of any one of Embodiments 338-339, wherein the alkylene is —CH₂—. 341. The method of any one of Embodiments 338-339, wherein the alkylene is —CH₂CH₂—. 342. The method of any one of Embodiments 338-339, wherein the alkylene is —CH₂CH₂CH₂—. 343. The method of Embodiment 284, wherein the aliphatic aldehyde is HO—CH₂—CH₂—CHO. 344. The method of Embodiment 285 or 343, wherein the aldol product is HO—CH₂—CH₂—CH(OH)—CH₂—C(O)—COOH or a salt thereof. 345. The method of any one of Embodiments 286 and 343-344, wherein the aldol-dehydration product is HO—CH₂—CH₂—CH═CH—C(O)—COOH or a salt thereof. 346. The method of any one of Embodiments 289 and 343-345, wherein the alkene reduction product is HO—CH₂—CH₂—CH₂—CH₂—C(O)—COOH or a salt thereof. 347. The method of any one of Embodiments 301 and 343-346, wherein the carbonyl reduction product is HO—CH₂—CH₂—CH₂—CH₂—CH(OH)—COOH or a salt thereof. 348. The method of any one of Embodiments 302 and 343-347, wherein a compound of formula P-5 or a salt thereof is HO—CH₂—CH₂—CH₂—CH₂—CH(OH)—CO—S—CoA or a salt thereof. 349. The method of any one of Embodiments 303 and 343-348, wherein a compound of formula P-6 or a salt thereof is HO—CH₂—CH₂—CH₂—CH═CH—CO—S—CoA or a salt thereof. 350. The method of any one of Embodiments 305 and 343-349, wherein a compound of formula P-7 or a salt thereof is HO—CH₂—CH₂—CH₂—CH₂—CH₂—CO—S-CoA or a salt thereof. 351. The method of any one of Embodiments 308 and 343-350, wherein a compound of formula P-8 or a salt thereof is HO—CH₂—CH₂—CH₂—CH₂—CH₂—CO—OH or a salt thereof. 352. The method of any one of Embodiments 310 and 343-351, wherein a compound of formula P-9 or a salt thereof is H—C(O)—CH₂—CH₂—CH₂—CH₂—CO—OH or a salt thereof. 353. The method of any one of Embodiments 312 and 343-352, wherein a compound of formula P-10 or a salt thereof is HO—CO—CH₂—CH₂—CH₂—CH₂—CO—OH or a salt thereof. 354. The method of any one of Embodiments 310 and 343-351, wherein a compound of formula P-9′ or a salt thereof is HO—CH₂—CH₂—CH₂—CH₂—CH₂—C(O)—H or a salt thereof. 355. The method of any one of Embodiments 312 and 343-351 and 354, wherein a compound of formula P-10′ or a salt thereof is HO—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—OH or a salt thereof. 356. The method of any one of Embodiments 317 and 343-346, wherein a compound of formula P-4′ or a salt thereof is HO—CH₂—CH₂—CH₂—CH₂—C(O)—H or a salt thereof. 357. The method of any one of Embodiments 317 and 343-346 and 356, wherein a compound of formula P-5′ or a salt thereof is HO—CH₂—CH₂—CH₂—CH₂—CH₂—OH or a salt thereof. 358. The method of any one of Embodiments 238-357, wherein a microbe comprises two or more biosynthesis polypeptides in the contacting steps. 359. The method of any one of Embodiments 238-358, comprising performing one or more contacting and/or conversion steps in one type of microbe, and one or more other contacting and/or conversion steps in another type of microbe. 360. The method of any one of Embodiments 238-359, comprising performing one or more contacting and/or conversion steps in one culture, and one or more other contacting and/or conversion steps in another culture. 361. The method of any one of Embodiments 238-359, comprising performing the contacting and/or conversion steps in a single culture. 362. The method of any one of Embodiments 238-361, wherein a microbe comprises all biosynthesis polypeptides recited in the contacting steps. 363. The method of Embodiment 362, comprising performing the contacting and/or conversion steps in a single culture. 364. The method of any one of the preceding Embodiments, wherein the product is produced at about or at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 250, or 300 g/L of culture. 365. The method of any one of the preceding Embodiments, wherein pyruvate utilization for a desired product is about or is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. 366. A preparation prepared by a method of any one of the preceding Embodiments. 367. A preparation of a compound of formula P-1, P-2, P-3, P-4, P-4′, P-5, P-5′, P-6, P-7, P-8, P-9, P-9′, P-10, or P-10′, or salt thereof, or a preparation prepared by a method of any one of the preceding Embodiments, which preparation is enriched for ¹⁴C isotope relative to that observed in a reference preparation of the compound, which reference preparation is prepared using fossil carbon source. 368. A preparation of a polyester, a polyester polyol, a polyurethane, nylon 6, nylon 6,6, a polycarbonate diol, diacrylate ester, or diglycidyl ether, which preparation is manufactured using a preparation prepared by a method of any one of the preceding claims. 369. The preparation of Embodiment 368, wherein the preparation is enriched for ¹⁴C isotope relative to that observed in a reference preparation of the compound, which reference preparation is prepared using fossil carbon source. 370. An nucleic acid encoding one or more biosynthesis polypeptides of any one of the preceding Embodiments. 371. The nucleic acid of Embodiment 370, wherein the nucleic acid differs from a natural nucleic acid which encodes the same biosynthesis polypeptide. 372. The nucleic acid of Embodiment 370 or 371, wherein the nucleic acid is optimized for expression in a microorganism. 373. An engineered microbe that produces an aldol product of an aliphatic aldehyde, the microbe comprising increased expression or activity of an aldol product biosynthesis polypeptide, wherein:

the carbonyl group of the aliphatic aldehyde is not conjugated to a alkenyl, alkynyl, or aromatic group;

the aldol product is a compound comprising an aldehyde or ketone group and a hydroxyl group attached to a beta-carbon of an aldehyde or ketone carbonyl group.

374. The microbe of Embodiment 373, wherein the aliphatic aldehyde is described in any one of Embodiments 238-363. 375. The microbe of Embodiment 373, wherein the aldol product is described in any one of Embodiments 238-363. 376. An engineered microbe that produces an aldol-dehydration product of an aliphatic aldehyde, the microbe comprising increased expression or activity of an aldol product biosynthesis polypeptide, an aldol-dehydration product biosynthesis polypeptide, a dehydration product biosynthesis polypeptide, or any combination thereof, wherein:

the carbonyl group of the aliphatic aldehyde is not conjugated to a alkenyl, alkynyl, or aromatic group; and

the aldol-dehydration product is a compound comprising an aldehyde or ketone group and a double bond conjugated with the aldehyde or ketone group.

377. The microbe of Embodiment 376, wherein the aliphatic aldehyde is described in any one of Embodiments 238-363. 378. The microbe of Embodiment 376, wherein the aldol-dehydration product is described in any one of Embodiments 238-363. 379. An engineered microbe that produces an alkene reduction product, the microbe comprising increased expression or activity of an alkene reduction product biosynthesis polypeptide, wherein:

the alkene comprises a double bond conjugated to a carbonyl group; and

a double bond conjugated to a carbonyl group in the alkene is reduced to a single bond to provide an alkene reduction product.

380. The microbe of Embodiment 379, wherein the alkene is described in any one of Embodiments 271-363. 381. The microbe of Embodiment 379, wherein the alkene reduction product is described in any one of Embodiments 238-363. 382. The microbe of any one of Embodiments 373-381, further comprising increased expression or activity of a biosynthesis polypeptide of any one of Embodiments 271-363. 383. A culture, comprising a microbe of any one of Embodiments 238-382, and one or more compounds independently of formulae P-1 to P-10, P-9′, P-10′, P-4′ or P-5′, or a salt thereof. 384. The culture of Embodiment 383, wherein one or more compounds are independently of higher levels compared to a reference culture of comparable microbes without the increased expression or activity of a biosynthesis polypeptide(s). 385. The culture of any one of Embodiments 383-384, wherein each of the compounds of formulae P-1 to P-10, P-9′, P-10′, P-4′ or P-5′, or a salt thereof is independently as described in any one of Embodiments 238-363. 386. A method, preparation, compound, organism, microorganism, culture or product as described herein.

EXAMPLES

The following examples are set forth below to illustrate the methods and results according to the disclosed subject matter. These examples are not intended to be inclusive of all aspects of the subject matter disclosed herein, but rather to illustrate certain representative methods and results. These examples are not intended to exclude equivalents and variations of the subject matter described herein which are apparent to one skilled in the art. Throughout the examples, sequences of enzymes or proteins are identified by their Uniprot ID or by their GenBank Accession Numbers (referred to as GenBank ID or GenBank Accession No.) or by their RefSeq ID. In case of Uniprot ID, the sequences are denoted by the primary (citable) accession number. RefSeq protein record represents non-redundant protein sequences within the NCBI database. Non-redundant protein records represent one exact sequence that has been observed once or many times in different strains or species.

Example 1: Enzymes that Catalyze Aldol-Dehydration Product Biosynthesis Using Aliphatic Aldehydes

It has not previously been demonstrated that trans-o-hydroxybenzylidenepyruvate hydratase-aldolases (EC 4.1.2.45)¹⁻⁵ or 4-(2-carboxyphenyl)-2-oxobut-3-enoate aldolases (E.C. 4.1.2.34; also referred to as trans-2′-carboxybenzalpyruvate hydratase-aldolases)⁶, referred cumulatively herein as hydratase-aldolases or Ads-Hyd, possess any aldol addition or aldol condensation activity on aliphatic aldehydes,¹⁻⁶ especially those without any unsaturation next to the aldehyde group.⁵ Instead, the aldol condensation activity of these enzymes has previously been limited to substrates wherein the newly formed unsaturation can be stabilized via conjugation to unsaturation present within the aldehyde substrate.¹⁻⁵ Examples of such aldehyde substrates include aromatic conjugated aldehydes such as benzaldehyde or alkenals (i.e., aliphatic aldehydes with double bonds between C2 and C3). It has been unexpectedly discovered that these hydratase-aldolases are capable of utilizing a number of aliphatic aldehydes, e.g., linear aldehydes of different carbon lengths and different functionalities as substrates and are able to provide aldol-dehydration products, without the intention to be limited by any theory, through carrying out both aldol addition and aldol condensation reactions with pyruvate as the donor (nucleophile) to give the corresponding 4-hydroxy-2-keto-carboxylic acids and 3,4-dehydro-2-keto-carboxylic acids respectively as products. Results for representative trans-o-hydroxybenzylidenepyruvate hydratase-aldolases (e.g., entries Ads-Hyd 2 & 9 in Table 1) and trans-2′-carboxybenzalpyruvate hydratase-aldolases (e.g., entry Ads-Hyd 3 in Table 1) are summarized in Table 1 for aldol-dehydration activity (both aldol addition and aldol condensation), wherein pyruvate is used as donor and acetaldehyde, propionaldehyde, and 3-hydroxy-propanal are used as acceptor aldehydes.

TABLE 1 Provided technologies are active toward various aldehydes. Uniprot ID or % Identitiy to following Activity on Different substrates Genbank or EC Ads-Hyd sequences 3-hydroxy- Ads-Hyd ID RefSeq ID Number A0A286PH18 P0A144 Q79EM8 Acetaldehyde Propanal propanal Ads-Hyd 1 D7C0E5 UA 93.6 ND ND + + + Ads-Hyd 2 P0A144 4.1.2.45 ND 100 38.3 + + + Ads-Hyd 3 Q79EM8 4.1.2.34 ND 38.3 100 + + NA Ads-Hyd 4 A0A0N0AHI8 UA 59.2 ND ND NT NT + Ads-Hyd 5 A0A0N1FRY3 UA 93.6 ND ND NT NT + Ads-Hyd 6 M3DYR1 UA 59 ND ND NT + + Ads-Hyd 7 W7SU48 UA 63 ND ND NT NT + Ads-Hyd 8 A0A286PH18 UA 100 13.7 17 NT + + Ads-Hyd 9 Q9X9Q6 4.1.2.45 ND 57 36.3 NT NT + Ads-Hyd 10 Q9WXH7 UA ND 55.6 36 NT + + Ads-Hyd 11 A4XDS1 UA ND 56 36.5 NT NT + Ads-Hyd 12 F2J6N9 UA ND 60.1 40.2 NT NT + Ads-Hyd 13 A0A063BFL5 UA ND 63.2 34.7 NT NT + Ads-Hyd 14 Q9ZHH6 UA ND 73.1 38.6 NT NT + Ads-Hyd 15 A0A0C1K853 UA ND 75.2 38.6 NT NT + Ads-Hyd 62 WP_034398482 UA ND 81.7 36.8 NT NT + Ads-Hyd 87 PYK12191 UA 50.4 ND ND NT NT + Ads-Hyd 96 A0A370X7D8 UA 55.8 ND ND NT NT + Ads-Hyd 104 WP_028222253 UA 56.1 ND ND NT NT + Ads-Hyd 65 F2J6L6 UA ND 59.8 39.8 NT NT + Ads-Hyd 89 A0A0N0L9F6 UA 54 ND ND NT NT + Ads-Hyd 97 A0A1G9YWG7 UA 56.6 ND ND NT NT + Ads-Hyd 68 A0A2U1BT09 UA ND 50.7 34.8 NT NT + Ads-Hyd 108 A0A244DHE8 UA 57.4 ND ND NT NT + Ads-Hyd 29 WP_107818191 UA ND 58.3 39.8 NT NT + Ads-Hyd 69 A0A023WZF9 UA ND 91.3 37.1 NT NT + Ads-Hyd 93 PYN48855 UA 49.3 ND ND NT NT + Ads-Hyd 98 A0A421PAQ6 UA 58.3 ND ND NT NT NA Ads-Hyd 99 WP_028217297 UA 56.7 ND ND NT NT + Ads-Hyd 100 WP_034507049 UA 56 ND ND NT NT NA Ads-Hyd 110 KMK64081.1 4.1.2.45 ND 56 36 + + + Ads-Hyd 111 WP_070028041.1 4.1.2.45 ND 35 35 NT NT + Ads-Hyd 112 KZL92449.1 4.1.1.4 40 ND ND NT NT + NT = Not tested; NA = Not active; + = active; UA = EC number is unassigned; ND = Actual value is not determined as sequence identity is too blow (~<25%)

Aldol addition and aldol condensation activity on aliphatic unconjugated aldehydes of different carbon lengths and functionalities by a subset of enzymes from Table 1 is summarized in Table 2, further demonstrating the versatility of unconjugated aldehyde substrates suitable for this reaction.

TABLE 2 Provided technologies are active toward various aldehydes. Aldol Addition Aldol Condensation Enzyme ID A B C D E F A B C D E F Ads-Hyd 1 Yes Yes NT NT Yes NT Yes Yes NT NT Yes NT Ads-Hyd 2 Yes Yes NT Yes Yes NT Yes Yes NT Yes Yes NT Ads-Hyd 108 Yes Yes NT NT Yes NT Yes Yes NT NT Yes NT Ads-Hyd 3 Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Ads-Hyd 8 Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Ads-Hyd 89 Yes Yes NT Yes Yes Yes Yes Yes NT Yes Yes Yes Ads-Hyd 110 Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Ads-Hyd 112 NT NT NT Yes Yes NT NT NT NT Yes Yes NT HpaI Yes Yes Yes Yes Yes Yes No No No No No No NT = Not tested; A = acetaldehyde; B = propionaldehyde; C = butyraldehyde; D = 2-hydroxy acetaldehyde; E = 3′-hydroxy-propanal; F = 4-hydroxy butyraldehyde

Among other things, the technologies provide high efficiency, e.g., in terms of product production rate, yield and/or utilization of substrates, e.g., pyruvate. In some embodiments, a biosynthesis polypeptide is about 50%, 100%, or 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100 fold or more active, as measured by production of comparable products under suitable conditions, compared to a relevant reference biosynthesis polypeptide. In some embodiments, the present disclosure provides highly efficient utilization of a substrate, e.g., pyruvate. In some embodiments, utilization of a substrate, e.g., pyruvate, is about or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In some embodiments, desired product concentration in a culture is about or is at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 g/L after a period of production time (e.g., 90 min). In some embodiments, a yield is about or at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 mg/L, or is about or at least about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.7, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 250, or 300 g/L. For example, Table 3 demonstrates dramatically improved efficiency of provided technologies compared to aldolases known previously to catalyze corresponding reactions: a representative trans-o-hydroxybenzylidenepyruvate hydratase-aldolase in Table 3 outperforms (e.g., >5 times activity) the other aldolases in terms of aldol addition activity on the tested substrates. Among other things, Table 4 demonstrated that Ads-Hyd enzymes can provide improved product yields as well as highly efficient utilization of substrate pyruvate compared to the comparative aldolases. This is particularly notable since pyruvate is a central metabolite and may be consumed by other reactions within a microorganism. As demonstrated herein, provided technologies comprising aldol-dehydration product biosynthesis polypeptides can effectively minimize pyruvate consumption in vivo by undesirable reactions, which is crucial to improve desired product yield in vivo.

TABLE 3 Provided technologies can provide high activity. Activity on Different substrates 3-hydroxy- Enzyme Type Enzyme ID Uniprot ID Acetaldehyde Propionaldehyde propanal aldolase yagE P75682 25000 NT NT aldolase nanA P0A6L4 25000 NT NT aldolase garL P23522 15000 NT NT aldolase eda P0A955 5000 NT NT aldolase dgoA Q6BF16 25000 NT NT aldolase Av-Ads M9YI86 NT 20000 NT aldolase Cg-Ads Q8NMD2 NT 45000 NT aldolase Cj-Ads A0A1J6QD42 NT 5000 NT aldolase Mt-Ads Q8RBI5 NT 5000 NT aldolase Ps-Ads A3LZU9 NT 25000 NT aldolase Sa-Ads Q4JC35 NT 30000 NT hydratase- Ads-Hyd 1 D7C0E5 270000 405000 NT aldolase aldolase Ss-Ads O54288 NT 25000 NT aldolase St-Ads F9VPG1 NT 25000 NT aldolase HpaI Q47098 15000 25000 NT NT = Not tested. For activity determination, pyruvate (20 g/L) was incubated with either acetaldehyde (40 g/L) or propionaldehyde (40 g/L) for 12 hr aerobically.

TABLE 4 Provided technologies can provide high yields and highly efficient substrate utilization. Product Formation % Pyruvate Used For Enzyme (g/L) After 90 mins Production Name Enzyme ID A B C A B C Sb Ads-Hyd Ads-Hyd >3 >3 + 25 57 NT 1 G12 Ads- Ads-Hyd NT NT + + NT Not Hyd 108 applicable Aldolase HpaI 0.2 0.7 +  1  4 Not applicable NT = Not tested; + = activity confirmed but not quantified; A = acetaldehyde; B = propionaldehyde; C = 3′-hydroxy-propanal

Although a few hydratase-aldolases have been categorized as belonging to EC 4.1.2.45 or EC 4.1.2.34 (see Table 5), most enzyme sequences reported in Table 1 and sequences identified by homology searches (using BLAST; see Tables 6-8) have not been assigned an EC number. Additionally, these enzymes have also been annonated in literature or databases (e.g., Uniprot) as acetoacetate decarboxylase or dihydrodipicolinate synthetase or simply as aldolases due to the similarity with these other classes of enzymes. For example, Ads-Hyd 8 enzyme is not annotated as a hydratase-aldolase and is annotated to be an acetoacetate decarboxylase (see Uniprot page for this sequence), when it functions as a hydratase-aldolase (see Table 1). Similarly, Ads-Hyd 11-13 enzymes have been annotated as dihydrodipicolinate synthetase, but they function as a hydratase-aldolase (see Table 1). It is expected that many hydratase-aldolase enzyme sequences are or will be annotated or inferred in public databases as belonging to acetoacetate decarboxylase or dihydrodipicolinate synthetase or aldolases and are not categorized to either belonging to EC 4.1.2.45 or EC 4.1.2.34. Thus, to identify hydratase-aldolase enzyme sequences, homology-based searches to hydratase-aldolase sequences were conducted, and the resultant enzymes were subsequently validated regarding their activity using methods described herein. An exemplary, homology-based search using (a) one sequence belonging to EC 4.1.2.34 (Ads-Hyd 3; results in Table 8); (b) one sequence belonging to an unassigned enzyme with extremely low homology to enzymes belonging to EC 4.1.2.34 and EC 4.1.2.45 (Ads-Hyd 8; results in Table 6) and (c) one sequence belonging to an unassigned enzyme show moderate homology to enzymes belonging to EC 4.1.2.34 and EC 4.1.2.45 (Ads-Hyd 10; results in Table 7) revealed >500 enzymes, some of which are listed in the tables below, and many of which upon testing were confirmed to be active for aldol addition and condensation (data in Table 1). For example, 13 sequences identified in Table 6 (see underlined sequences in Table 6 with data for those sequence in Table 1), and 11 sequences identified in Table 7 (see underlined sequences in Table 7 with data for those sequence in Table 1) were confirmed to be functional Ads-Hyd enzymes. Among other things, the present disclosure demonstrated that Ads-Hyd 112, which is classified as belonging to E.C 4.1.1.4 and annontated as an acetetoacetate decarboxylase, was also found to catalyze aldol addition and aldol condensation reactions with a number of different aldehydes (Table 2). In some embodiments, enzymes annotated as acetoacetate decarboxylases as well as those belonging to E.C 4.1.1.4 are useful for catalyzing aldol condensation and addition reactions as well. Enzymes with identities ranging from as low as 35% (Ads-Hyd 68 in Table 1), 38% (Ads-Hyd 3 in Table 1) and 49% (Ads-Hyd 93 in Table 1) to Ads-Hyd 3 belonging to EC 4.1.2.34, Ads-Hyd 2 belonging to EC 4.1.2.45, and Ads-Hyd 8 enzymes respectively, were confirmed to have hydratase-aldolase activity.

TABLE 5 Certain biosynthesis polypeptides. EC Uniprot ID Genbank ID Number Protein names Q9X9Q6 AAD45417.1 4.1.2.45 Trans-O-hydroxybenzylidenepyruvate hydratase-aldolase (2′- hydroxybenzalpyruvate aldolase) P0A144 AAB62713.1 4.1.2.45 Trans-O-hydroxybenzylidenepyruvate hydratase-aldolase (2′- hydroxybenzalpyruvate aldolase) P0A142 BAA12246.1 4.1.2.45 Trans-O-hydroxybenzylidenepyruvate hydratase-aldolase (2′- hydroxybenzalpyruvate aldolase) Q79EM8 BAA23263.1 4.1.2.34 Trans-2′-carboxybenzalpyruvate hydratase- aldolase Q51947 AAA66357.1 4.1.2.45 Trans-O-hydroxybenzylidenepyruvate hydratase-aldolase (2′- hydroxybenzalpyruvate aldolase) P0A143 AAA16132.1 4.1.2.45 Trans-O-hydroxybenzylidenepyruvate hydratase-aldolase (2′- hydroxybenzalpyruvate aldolase) A0A0J5Q5D8 KMK64081.1 4.1.2.45 Dihydrodipicolinate synthase/N- acetylneuraminate lyase A0A1Y5PJE4 SBS78822.1 4.1.2.34 Trans-2′-carboxybenzalpyruvate hydratase- aldolase A0A2H5YJ14 GBD13407.1 4.1.2.34 Trans-2′-carboxybenzalpyruvate hydratase- aldolase A0A1V6C3X5 OQB71622.1 4.1.2.45 Trans-O-hydroxybenzylidenepyruvate hydratase-aldolase (2′- hydroxybenzalpyruvate aldolase) A0A2H5YYR5 GBD18589.1 4.1.2.45 Trans-O-hydroxybenzylidenepyruvate hydratase-aldolase (2′- hydroxybenzalpyruvate aldolase) A0A2H5VLK1 GBC77546.1 4.1.2.45 Trans-O-hydroxybenzylidenepyruvate hydratase-aldolase (2′- hydroxybenzalpyruvate aldolase) A0A1K2FZU3 SFY52690.1 4.1.2.45 Trans-O-hydroxybenzylidenepyruvate hydratase-aldolase (2′- hydroxybenzalpyruvate aldolase) A0A2H5W1Y6 GBC82821.1 4.1.2.45 Trans-O-hydroxybenzylidenepyruvate hydratase-aldolase (2′- hydroxybenzalpyruvate aldolase)

TABLE 6 Certain biosynthesis polypeptides - enzymes that show homology to Ads-Hyd 8. Genbank ID (Enzyme ID if verified) Protein names KZL92449.1 (Ads-Hyd 112) Acetoacetate decarboxylase (EC 4.1.1.4) AOS64057.1 Acetoacetate decarboxylase (ADC) (EC 4.1.1.4) GBC87126.1 Acetoacetate decarboxylase (EC 4.1.1.4) AKL97316.1 Acetoacetate decarboxylase (EC 4.1.1.4) PZG 10242.1 Acetoacetate decarboxylase AEB44722.1 Acetoacetate decarboxylase ABG04000.1 Acetoacetate decarboxylase KPH00942.1 (Ads-Hyd 89) Acetoacetate decarboxylase EMF26762.1 (Ads-Hyd 6) Acetoacetate decarboxylase PVY06388.1 Acetoacetate decarboxylase AOJ06649.1 Acetoacetate decarboxylase KOX08160.1 (Ads-Hyd 4) Acetoacetate decarboxylase OPG13060.1 Enduracididine biosynthesis enzyme MppR SDQ34954.1 Acetoacetate decarboxylase OIJ66442.1 Acetoacetate decarboxylase GCE00545.1 Acetoacetate decarboxylase ACK51122.1 Acetoacetate decarboxylase PJJ78777.1 Acetoacetate decarboxylase GAU76561.1 Acetoacetate decarboxylase SEM36970.1 Acetoacetate decarboxylase REK87553.1 Enduracididine biosynthesis enzyme MppR KPI02092.1 (Ads-Hyd 5) Acetoacetate decarboxylase KPC94750.1 Acetoacetate decarboxylase (Fragment) AEF90707.1 Acetoacetate decarboxylase OPC78676.1 Acetoacetate decarboxylase OPY57828.1 Acetoacetate decarboxylase KUL75432.1 Acetoacetate decarboxylase OEV06324.1 Acetoacetate decarboxylase PVX87320.1 Acetoacetate decarboxylase PIG16285.1 Acetoacetate decarboxylase POR47715.1 Acetoacetate decarboxylase SFH06339.1 Acetoacetate decarboxylase KUM42217.1 Acetoacetate decarboxylase PZT77592.1 Acetoacetate decarboxylase KYC38950.1 Acetoacetate decarboxylase RKS77249.1 Acetoacetate decarboxylase OIJ92678.1 Acetoacetate decarboxylase BAU27837.1 Acetoacetate decarboxylase QAV71426.1 Acetoacetate decarboxylase PQZ48703.1 Uncharacterized protein EXU61971.1 Acetoacetate decarboxylase SHN38127.1 Acetoacetate decarboxylase KGT73177.1 Acetoacetate decarboxylase SIO29145.1 Acetoacetate decarboxylase KGT73210.1 Acetoacetate decarboxylase SIO27946.1 Acetoacetate decarboxylase OSJ25700.1 Acetoacetate decarboxylase RMD31380.1 Acetoacetate decarboxylase SIO53681.1 Acetoacetate decarboxylase RFU48568.1 Acetoacetate decarboxylase OSJ25816.1 Acetoacetate decarboxylase HCV33217.1 Acetoacetate decarboxylase KPD20047.1 Acetoacetate decarboxylase OFW57075.1 Uncharacterized protein HCW00147.1 Acetoacetate decarboxylase EIM94241.1 Acetoacetate decarboxylase OYV58956.1 Acetoacetate decarboxylase REK15702.1 Acetoacetate decarboxylase MBV14559.1 Acetoacetate decarboxylase HAN36693.1 Acetoacetate decarboxylase HAP74745.1 Acetoacetate decarboxylase PYR38950.1 Acetoacetate decarboxylase (Fragment) PYR49219.1 Acetoacetate decarboxylase PTB41031.1 Uncharacterized protein EHK39542.1 Uncharacterized protein SYX90497.1 Acetoacetate decarboxylase RKN45560.1 Acetoacetate decarboxylase KJC40693.1 Uncharacterized protein RKR91249.1 Acetoacetate decarboxylase EJL77881.1 Acetoacetate decarboxylase PIG41119.1 Acetoacetate decarboxylase KJC40569.1 Acetoacetate decarboxylase KGF80061.1 Acetoacetate decarboxylase SON57276.1 Acetoacetate decarboxylase (ADC) KYO55945.1 Acetoacetate decarboxylase RFC69939.1 Acetoacetate decarboxylase RPE56489.1 Acetoacetate decarboxylase SFQ35591.1 Acetoacetate decarboxylase SCD72996.1 Acetoacetate decarboxylase RQO46864.1 Acetoacetate decarboxylase RLK57997.1 Enduracididine biosynthesis enzyme MppR ACZ90180.1 Acetoacetate decarboxylase GCD42233.1 Uncharacterized protein PIF96550.1 Enduracididine biosynthesis enzyme MppR PBC93106.1 Acetoacetate decarboxylase SIO44972.1 Acetoacetate decarboxylase OYD73530.1 Acetoacetate decarboxylase SEC28728.1 Enduracididine biosynthesis enzyme MppR RFC78087.1 Acetoacetate decarboxylase PWC35104.1 Acetoacetate decarboxylase AWL33917.1 Enduracididine biosynthesis enzyme MppR SED37560.1 Acetoacetate decarboxylase KOG37070.1 Acetoacetate decarboxylase SDJ19059.1 Enduracididine biosynthesis enzyme MppR PHX81843.1 Acetoacetate decarboxylase MBJ31847.1 Acetoacetate decarboxylase RPJ15459.1 Acetoacetate decarboxylase ABD65946.1 Enduracididine biosynthesis enzyme MppR RSM78635.1 Acetoacetate decarboxylase RSM86524.1 Acetoacetate decarboxylase AUG07753.1 Acetoacetate decarboxylase SHG60447.1 Acetoacetate decarboxylase SMC73048.1 Acetoacetate decarboxylase PKR44685.1 Enduracididine biosynthesis enzyme MppR AUC95510.1 Acetoacetate decarboxylase SUZ73052.1 Uncharacterized protein (Fragment) SNS52433.1 Acetoacetate decarboxylase (ADC) MMZ55024.1 Acetoacetate decarboxylase MNQ33472.1 Acetoacetate decarboxylase KJC46837.1 Acetoacetate decarboxylase SDL38666.1 Acetoacetate decarboxylase ONI74756.1 Acetoacetate decarboxylase SOD30619.1 Acetoacetate decarboxylase KJC46838.1 Acetoacetate decarboxylase RUL62263.1 Acetoacetate decarboxylase RMI93268.1 (Ads-Hyd 98) Acetoacetate decarboxylase RKR21285.1 Acetoacetate decarboxylase SDK87733.1 Acetoacetate decarboxylase PZS29802.1 Acetoacetate decarboxylase AAU34211.1 Uncharacterized protein CNE94443.1 Acetoacetate decarboxylase CDR14781.1 Acetoacetate decarboxylase OGI63453.1 Acetoacetate decarboxylase SDW59396.1 Enduracididine biosynthesis enzyme MppR MBE40108.1 Acetoacetate decarboxylase RPI20925.1 Acetoacetate decarboxylase AVZ77933.1 Acetoacetate decarboxylase CRK83612.1 Acetoacetate decarboxylase AOP51678.1 Enduracididine biosynthesis enzyme MppR KJC56449.1 Uncharacterized protein POX38729.1 Acetoacetate decarboxylase RDS84232.1 (Ads-Hyd 96) Acetoacetate decarboxylase ABK52869.1 Acetoacetate decarboxylase ERI08645.1 Putative acetoacetate decarboxylase SED02700.1 Acetoacetate decarboxylase SED57674.1 Acetoacetate decarboxylase AJQ29697.1 Acetoacetate decarboxylase AUS77184.1 Enduracididine biosynthesis enzyme MppR OEV05744.1 Enduracididine biosynthesis enzyme MppR SHJ82744.1 Acetoacetate decarboxylase (ADC) PDQ21702.1 Acetoacetate decarboxylase MBF06178.1 Acetoacetate decarboxylase SDI62088.1 Acetoacetate decarboxylase SES42580.1 Enduracididine biosynthesis enzyme MppR OAN53209.1 Acetoacetate decarboxylase CUU19651.1 Acetoacetate decarboxylase CDS PIG70517.1 Acetoacetate decarboxylase GAT80125.1 Acetoacetate decarboxylase RMI45923.1 Acetoacetate decarboxylase RFS83293.1 Acetoacetate decarboxylase RUL90134.1 Enduracididine biosynthesis enzyme MppR CEH29276.1 Putative acetoacetate decarboxylase KJC56043.1 Acetoacetate decarboxylase KJC56044.1 Acetoacetate decarboxylase AWE54161.1 Acetoacetate decarboxylase ADI03636.1 (Ads-Hyd 1) Acetoacetate decarboxylase GAT84669.1 Acetoacetate decarboxylase RUQ72183.1 Acetoacetate decarboxylase RSN12399.1 Acetoacetate decarboxylase RKD49684.1 Acetoacetate decarboxylase RKR34606.1 Acetoacetate decarboxylase PIG06713.1 Acetoacetate decarboxylase ROQ34846.1 Enduracididine biosynthesis enzyme MppR KXU84461.1 Acetoacetate decarboxylase OUL77098.1 (Ads-Hyd 108) Acetoacetate decarboxylase PYK12191.1 (Ads-Hyd 87) Acetoacetate decarboxylase RUL72479.1 Acetoacetate decarboxylase PWK86305.1 Enduracididine biosynthesis enzyme MppR GCD34260.1 Uncharacterized protein SOE90358.1 Acetoacetate decarboxylase SDG84621.1 Enduracididine biosynthesis enzyme MppR EWM12399.1 (Ads-Hyd 7) Acetoacetate decarboxylase SDG23054.1 Acetoacetate decarboxylase AFK55453.1 Uncharacterized protein AUT62680.1 Acetoacetate decarboxylase RPE37958.1 Acetoacetate decarboxylase EWM12653.1 Acetoacetate decarboxylase RSN04866.1 Enduracididine biosynthesis enzyme MppR KQV82686.1 Acetoacetate decarboxylase RKF38182.1 Acetoacetate decarboxylase REE27044.1 Acetoacetate decarboxylase PJN40277.1 Enduracididine biosynthesis enzyme MppR SDN12921.1 Acetoacetate decarboxylase PYG36199.1 Acetoacetate decarboxylase RKQ65112.1 Acetoacetate decarboxylase SDN12891.1 (Ads-Hyd 97) Acetoacetate decarboxylase EIW19392.1 Acetoacetate decarboxylase RSN99590.1 Acetoacetate decarboxylase PON28167.1 Uncharacterized protein PNP43262.1 Uncharacterized protein PON20078.1 Uncharacterized protein AEM85455.1 Acetoacetate decarboxylase AOT70611.1 Acetoacetate decarboxylase OPF83246.1 Acetoacetate decarboxylase PYN48855.1 (Ads-Hyd 93) Acetoacetate decarboxylase SFH92960.1 Acetoacetate decarboxylase SME92731.1 Acetoacetate decarboxylase RKQ67404.1 Acetoacetate decarboxylase RAK24761.1 Acetoacetate decarboxylase ALV48823.1 Acetoacetate decarboxylase SHG57166.1 Acetoacetate decarboxylase SHI09865.1 Acetoacetate decarboxylase RLV76922.1 Acetoacetate decarboxylase SHG57190.1 Acetoacetate decarboxylase KXU84652.1 Acetoacetate decarboxylase SIO27627.1 Acetoacetate decarboxylase AXQ55553.1 Enduracididine biosynthesis enzyme MppR AOJ04944.1 Acetoacetate decarboxylase ARH95437.1 Enduracididine biosynthesis enzyme MppR REH48625.1 Acetoacetate decarboxylase RLJ42250.1 Acetoacetate decarboxylase SHN71285.1 Acetoacetate decarboxylase SHN71288.1 Acetoacetate decarboxylase SHI09851.1 Acetoacetate decarboxylase SHN71296.1 Acetoacetate decarboxylase SIO27636.1 Acetoacetate decarboxylase REH35177.1 Acetoacetate decarboxylase SOE93021.1 Acetoacetate decarboxylase ALO91482.1 Acetoacetate decarboxylase AKJ70148.1 Acetoacetate decarboxylase EJL71335.1 Acetoacetate decarboxylase KMS76577.1 Acetoacetate decarboxylase SAL51447.1 Acetoacetate decarboxylase MBA77131.1 Acetoacetate decarboxylase MAM76769.1 Acetoacetate decarboxylase AXL50798.1 Acetoacetate decarboxylase SOE99541.1 Acetoacetate decarboxylase PIF38354.1 Acetoacetate decarboxylase GAX58847.1 Acetoacetate decarboxylase SFN30008.1 Acetoacetate decarboxylase KUL58863.1 Enduracididine biosynthesis enzyme MppR KOG74850.1 Acetoacetate decarboxylase AEY87061.1 Acetoacetate decarboxylase RDS66140.1 Acetoacetate decarboxylase ONI72521.1 Acetoacetate decarboxylase AHH95455.1 Carboxy-lyase SOE19480.1 Acetoacetate decarboxylase (ADC) ROO80377.1 Acetoacetate decarboxylase SAL27032.1 Acetoacetate decarboxylase HAM27991.1 Acetoacetate decarboxylase KDN75868.1 Acetoacetate decarboxylase AEW99245.1 Uncharacterized protein AAR35773.1 Acetoacetate decarboxylase family protein PMR61960.1 Acetoacetate decarboxylase OXL32653.1 Acetoacetate decarboxylase KUN27737.1 Acetoacetate decarboxylase EPR75769.1 Acetoacetate decarboxylase SFT90048.1 Acetoacetate decarboxylase RFU39638.1 Acetoacetate decarboxylase SMG22616.1 Acetoacetate decarboxylase

TABLE 7 Certain biosynthesis polypeptides - enzymes that show homology to Ads-Hyd 10. Genbank ID (Enzyme ID) Protein names SBS78822.1 Trans-2′-carboxybenzalpyruvate hydratase-aldolase (EC 4.1.2.34) GBD13407.1 Trans-2′-carboxybenzalpyruvate hydratase-aldolase (EC 4.1.2.34) BAA23263.1 (Ads-Hyd 3) Trans-2′-carboxybenzalpyruvate hydratase-aldolase (EC 4.1.2.34) AAD45417.1 (Ads-Hyd 9) Trans-O-hydroxybenzylidenepyruvate hydratase-aldolase (EC 4.1.2.45) AAA16132.1 Trans-O-hydroxybenzylidenepyruvate hydratase-aldolase (EC 4.1.2.45) BAA12246.1 Trans-O-hydroxybenzylidenepyruvate hydratase-aldolase (EC 4.1.2.45) AAB62713.1 (Ads-Hyd 2) Trans-O-hydroxybenzylidenepyruvate hydratase-aldolase (EC 4.1.2.45) AAA66357.1 Trans-O-hydroxybenzylidenepyruvate hydratase-aldolase (EC 4.1.2.45) KMK64081.1 Dihydrodipicolinate synthase/N-acetylneuraminate lyase (EC 4.1.2.45) GBD18589.1 Trans-O-hydroxybenzylidenepyruvate hydratase-aldolase (EC 4.1.2.45) GBC82821.1 Trans-O-hydroxybenzylidenepyruvate hydratase-aldolase (EC 4.1.2.45) ART89851.1 4-hydroxy-tetrahydrodipicolinate synthase (EC 4.3.3.7) SJM52860.1 4-hydroxy-tetrahydrodipicolinate synthase (EC 4.3.3.7) ART58441.1 Aldolase BAA76332.1 (Ads-Hyd 10) Hydratase-aldolase AEF88788.1 Dihydrodipicolinate synthetase ART51183.1 Aldolase KLU36881.1 Aldolase AKM12047.1 Aldolase CCA93880.1 Dihydrodipicolinate synthetase EZP70093.1 Putative 2-hydroxy-benzylpyruvate aldolase EHJ58034.1 Putative 2-hydroxy-benzylpyruvate aldolase ART40746.1 L352 ATW03328.1 Aldolase CCA92467.1 Dihydrodipicolinate synthetase ABM79813.1 Aldolase (Hydratase-aldolase) BAC65452.1 Putative 2-hydroxy-benzylpyruvate aldolase GAM16817.1 Hydratase-aldolase PBN41471.1 Trans-o-hydroxybenzylidenepyruvate hydratase-aldolase OWQ92810.1 Aldolase SHN54758.1 Dihydrodipicolinate synthase/N-acetylneuraminate lyase KDA01194.1 Dihydrodipicolinate synthetase KJS38380.1 Aldolase AKQ42951.1 1,2-dihydroxybenzylpyruvate aldolase PNU02635.1 Aldolase EJU12841.1 1,2-dihydroxybenzylpyruvate aldolase OAP30848.1 Aldolase ETI62764.1 Aldolase KKW89821.1 Aldolase PNQ03402.1 Aldolase AGZ63484.1 NahE PKB13561.1 Dihydrodipicolinate synthase/N-acetylneuraminate lyase PEQ10932.1 Aldolase AYO76044.1 Aldolase ABP64082.1 (Ads-Hyd 11) Dihydrodipicolinate synthetase KHS42353.1 Dihydrodipicolinate synthetase AAD03976.1 1,2-dihydroxybenzylpyruvate aldolase KTE40403.1 Aldolase KTE22766.1 Aldolase RJG53082.1 Aldolase PQM29276.1 Aldolase KTE33221.1 Aldolase KGB52059.1 Putative 2-hydroxy-benzylpyruvate aldolase ART37867.1 F474 ODU68266.1 Aldolase (Fragment) PXV63448.1 Trans-o-hydroxybenzylidenepyruvate hydratase-aldolase AJP47897.1 Aldolase ADZ72499.1 (Ads-Hyd 65) Dihydrodipicolinate synthetase AER08042.1 Hydratase-aldolase EIF28466.1 Dihydrodipicolinate synthase/N-acetylneuraminate lyase ALE55172.1 Aldolase OWJ56339.1 Aldolase PJJ06708.1 Trans-o-hydroxybenzylidenepyruvate hydratase-aldolase AKM10279.1 Aldolase ART40122.1 K159 ART38154.1 F222 PWJ76345.1 Trans-o-hydroxybenzylidenepyruvate hydratase-aldolase PTQ67744.1 Trans-o-hydroxybenzylidenepyruvate hydratase-aldolase KGB81035.1 Aldolase PTQ65074.1 Trans-o-hydroxybenzylidenepyruvate hydratase-aldolase ADZ72522.1 (Ads-Hyd 12) Dihydrodipicolinate synthetase ALG92322.1 Aldolase KEP68746.1 Aldolase AMM86059.1 Aldolase MAM12073.1 Aldolase EIT71336.1 Dihydrodipicolinate synthetase AEF05081.1 Dihydrodipicolinate synthetase PAL23311.1 Aldolase PWJ76353.1 Trans-o-hydroxybenzylidenepyruvate hydratase-aldolase RVT39492.1 Aldolase SEP74235.1 Trans-o-hydroxybenzylidenepyruvate hydratase-aldolase BAA20397.1 Hydratase-aldolase AAL07266.1 2-hydroxybenzalpyruvate aldolase ETI60157.1 Aldolase ART36295.1 C905 BAF34962.1 Trans-o-hydrobenzylidenepyruvate hydratase aldolase BAF34972.1 Trans-o-hydrobenzylidenepyruvate hydratase aldolase AAP44192.1 1,2-dihydroxybenzyl pyruvate aldolase EXF90974.1 Aldolase OPK08859.1 Trans-o-hydroxybenzylidenepyruvate hydratase-aldolase APV43293.1 Aldolase (Trans-o-hydroxybenzylidenepyruvate hydratase-aldolase) AAO64280.1 Trans-o-hydroxybenzylidenepyruvate hydratase-aldolase ALC77286.1 Trans-O-hydroxybenzylidenepyruvate hydratase-aldolase ACQ63497.1 Trans-o-hydroxybenzylidenepyruvate hydratase-aldolase ASW04047.1 Aldolase KKC26031.1 Aldolase AEV45882.1 Trans-o-hydroxybenzylidenepyruvate hydratase-aldolase NahE BAE92162.1 Trans-o-hydroxybenzylidenepyruvate hydratase-aldolase NahE BAF30942.1 Trans-ohydrobenzylidenepyruvate hydratase aldolase APP18116.1 Trans-o-hydroxybenzylidenepyruvate hydratase-aldolase AEV41420.1 Trans-o-hydroxybenzylidenepyruvate hydratase-aldolase AAD02141.1 1, 2-dihydroxybenzylpyruvate aldolase OCX93212.1 Aldolase EPL61966.1 Trans-o-hydroxybenzylidenepyruvate hydratase-aldolase AFM32586.1 Trans-o-hydroxybenzylidenepyruvate hydratase-aldolase AAD12616.1 Trans-o-hydroxybenzylidenepyruvate hydratase-aldolase MAS13884.1 Aldolase EWC41257.1 Aldolase AHY45199.1 (Ads-Hyd 69) Aldolase AJE45 066.1 Dihydrodipicolinate synthetase VBB16389.1 Aldolase AAZ93394.1 Dihydrodipicolinate synthetase (Trans-o-hydroxybenzylidenepyruvate hydratase-aldolase) SAL31848.1 Dihydrodipicolinate synthetase family protein ALE55136.1 Aldolase OWJ56143.1 Aldolase AAD09869.1 (Ads-hyd 14) Hydratase/aldolase PhnE ACT53260.1 Hydratase/aldolase ANI13636.1 Aldolase EZQ14085.1 Aldolase PRF53899.1 Aldolase EHJ59545.1 Hydratase-aldolase ODU66836.1 Aldolase AZI70977.1 1,2-dihydroxybenzylpyruvate aldolase (Trans-o-hydroxybenzylidenepyruvate hydratase-aldolase) KIC79255.1 (Ads-Hyd 15) Aldolase AMK37583.1 Trans-o-hydroxybenzylidenepyruvate hydratase-aldolase KGH10186.1 Aldolase PHR55511.1 Aldolase RAK18497.1 Trans-o-hydroxybenzylidenepyruvate hydratase-aldolase EHJ59565.1 2-hydroxybenzalpyruvate aldolase PVY51792.1 (Ads-Hyd 68) Dihydrodipicolinate synthase/N-acetylneuraminate lyase KDB08187.1 (Ads-Hyd 13) Dihydrodipicolinate synthetase APP18130.1 Hydratase-aldolase EHJ59532.1 Hydratase/aldolase EIE49938.1 Trans-o-hydroxybenzylidenepyruvate hydratase-aldolase NahE KHK92942.1 Aldolase ART39436.1 J508 RSM40400.1 Aldolase HAC32985.1 Aldolase SED12223.1 Dihydrodipicolinate synthase/N-acetylneuraminate lyase ART36910.1 D219 HCO44328.1 Aldolase OUR88246.1 Aldolase ANX02865.1 Aldolase PCI67543.1 Aldolase SHJ43395.1 Trans-o-hydroxybenzylidenepyruvate hydratase-aldolase AGS39599.1 Dihydrodipicolinate synthase/N-acetylneuraminate lyase MBG95280.1 Aldolase AFT67194.1 Dihydrodipicolinate synthetase PHS71704.1 Aldolase HAI96648.1 Aldolase EHJ59569.1 Dihydrodipicolinate synthetase AIN43768.1 Hydratase-aldolase (Fragment) ART35398.1 A220 SDM13008.1 Trans-o-hydroxybenzylidenepyruvate hydratase-aldolase SDG98718.1 Trans-o-hydroxybenzylidenepyruvate hydratase-aldolase EKX84573.1 Trans-o-hydroxybenzylidenepyruvate hydratase-aldolase NahE RTL66015.1 Aldolase KPK20478.1 Uncharacterized protein SEH64089.1 Dihydrodipicolinate synthase/N-acetylneuraminate lyase ART37041.1 D408 PYC47978.1 Aldolase OUS22376.1 Uncharacterized protein ANX03747.1 Uncharacterized protein KDE97295.1 Aldolase OPX10770.1 Uncharacterized protein ODQ85801.1 Aldolase ORB11495.1 Aldolase ORA58811.1 Aldolase ABL90862.1 Dihydrodipicolinate synthetase ADT96876.1 Dihydrodipicolinate synthase/N-acetylneuraminate lyase ABM11316.1 Dihydrodipicolinate synthetase BBA72532.1 Dihydrodipicolinate synthetase GAT12856.1 Dihydrodipicolinate synthetase ARV80195.1 Aldolase (Dihydrodipicolinate synthase/N-acetylneuraminate lyase) ABP43078.1 Dihydrodipicolinate synthetase AKK27886.1 Aldolase SEH58270.1 4-(2-carboxyphenyl)-2-oxobut-3-enoate aldolase APE19406.1 Aldolase AAT51742.1 PhdJ BBA72542.1 Dihydrodipicolinate synthetase BBA72825.1 Dihydrodipicolinate synthetase AEV73682.1 Dihydrodipicolinate synthase/N-acetylneuraminate lyase ORB61988.1 Aldolase RDH74327.1 Aldolase ACN38282.1 Trans-2′-carboxybenzalpyruvate hydratase-aldolase KLU36867.1 Aldolase OUS03890.1 Uncharacterized protein ORW27057.1 Uncharacterized protein OAR05193.1 4-hydroxy-tetrahydrodipicolinate synthase (Aldolase) CQD18686.1 Dihydrodipicolinate synthetase ORB04914.1 Uncharacterized protein AJP48436.1 Uncharacterized protein ACM06757.1 Aldolase HCO44883.1 Aldolase ANX04975.1 Uncharacterized protein SPM40709.1 Dihydrodipicolinate synthase/N-acetylneuraminate lyase SPM34880.1 Dihydrodipicolinate synthase/N-acetylneuraminate lyase OLT42115.1 Aldolase HAC33263.1 Aldolase RFU95674.1 Dihydrodipicolinate synthetase OGQ80071.1 Uncharacterized protein SFB53516.1 Dihydrodipicolinate synthase/N-acetylneuraminate lyase PVY51809.1 Dihydrodipicolinate synthase/N-acetylneuraminate lyase PVY51803.1 Dihydrodipicolinate synthase/N-acetylneuraminate lyase ORB07056.1 Aldolase PVY51800.1 Dihydrodipicolinate synthase/N-acetylneuraminate lyase RIA44335.1 Dihydrodipicolinate synthase/N-acetylneuraminate lyase EHJ59573.1 Uncharacterized protein PVY51825.1 Dihydrodipicolinate synthase/N-acetylneuraminate lyase AIJ21944.1 Putative aldolase ORB38363.1 Aldolase AWK75959.1 Aldolase EID78824.1 Putative aldolase NarC ACV96860.1 Putative aldolase HAC33092.1 Aldolase AKM10259.1 Uncharacterized protein ART40134.1 K171 ELB89137.1 Putative aldolase NarC BAH47216.1 Putative aldolase NarC AAR05117.1 Putative aldolase EKT84398.1 Putative aldolase NarC KDE09923.1 Aldolase BAE53379.1 Aldolase AAR05109.1 Putative aldolase AQW45620.1 Putative aldolase API60260.1 Uncharacterized protein RLA50226.1 Aldolase OUZ12202.1 Aldolase RLV57233.1 Aldolase BAA94711.1 Hydratase-aldolase AFC42746.1 Dihydrodipicolinate synthetase ASW94610.1 Aldolase ORW23722.1 Aldolase ORB75698.1 Aldolase AAG53397.1 1,2-dihydroxybenzylpyruvate aldolase 2 (Fragment) CRL08851.1 2-carboxybenzalpyruvate hydratase aldolase OSC27070.1 Aldolase RKO19521.1 Aldolase ADX75098.1 Dihydrodipicolinate synthase/N-acetylneuraminate lyase RTL66022.1 Dihydrodipicolinate synthetase AAG53396.1 1,2-dihydroxybenzylpyruvate aldolase 1 (Fragment) ADK82461.1 Dihydrodipicolinate synthetase OLT33718.1 Aldolase ADX73348.1 Dihydrodipicolinate synthase/N-acetylneuraminate lyase PSQ18743.1 Aldolase APA86915.1 Aldolase RAW15463.1 Aldolase AYY15006.1 Aldolase SEH58300.1 Hydratase-aldolase ORB22843.1 Aldolase

TABLE 8 Certain biosynthesis polypeptides - enzymes that show homology to Ads-Hyd 3. Genbank ID Protein names WP_013601270.1 aldolase [Pseudarthrobacter phenanthrenivorans] WP_013602982.1 aldolase [Pseudarthrobacter phenanthrenivorans] WP_127127049.1 aldolase [Georgenia sp. SYP-B2076] WP_075839590.1 aldolase [Rhodococcus sp. CUA-806] WP_086725852.1 aldolase [Streptomyces carpinensis] WP_137144035.1 aldolase [Mycolicibacterium sp. CR10] WP_047330709.1 aldolase [Mycobacterium sp. EPa45] WP_011559036.1 MULTISPECIES: aldolase [Mycobacteriaceae] WP_036349078.1 aldolase [Mycolicibacterium aromaticivorans] RTL66015.1 aldolase [Pseudonocardiaceae bacterium] WP_087139803.1 aldolase [Mycobacterium chimaera] WP_011777788.1 aldolase [Mycolicibacterium vanbaalenii] WP_011891552.1 aldolase [Mycolicibacterium gilvum] WP_069416983.1 aldolase [Mycolicibacterium flavescens] WP_083043896.1 aldolase [Mycolicibacterium elephantis] WP_083410401.1 aldolase [Mycolicibacterium rutilum] BBA72542.1 dihydrodipicolinate synthetase [Mycobacterium sp. PO1] WP_042910008.1 MULTISPECIES: aldolase [Mycobacterium avium complex (MAC)] WP_067396827.1 aldolase [Mycolicibacterium novocastrense] WP_083128714.1 aldolase [Mycolicibacterium tusciae] AAT51742.1 PhdJ [Mycolicibacterium vanbaalenii PYR-1] WP_114740710.1 aldolase [Mycolicibacterium moriokaense] WP_071950246.1 aldolase [Mycobacterium sp. WY10] GAT12856.1 dihydrodipicolinate synthetase [Mycolicibacterium novocastrense] WP_094286221.1 aldolase [Mycobacterium lehmannii] BBA72532.1 dihydrodipicolinate synthetase [Mycobacterium sp. PO1] WP_041303477.1 aldolase [Mycolicibacterium rhodesiae] AEV73682.1 dihydrodipicolinate synthase/N-acetylneuraminate lyase [Mycolicibacterium rhodesiae NBB3] 6DAQ_A Chain A, PhdJ [Mycolicibacterium vanbaalenii] WP_099039075.1 aldolase [Mycobacterium sp. CECT 8778] ACN38282.1 trans-2′-carboxybenzalpyruvate hydratase-aldolase [Mycobacterium sp. CH1] CRL08851.1 2-carboxybenzalpyruvate hydratase aldolase [Mycobacterium sp. 6PY1] WP_096699239.1 aldolase [Polaromonas sp. AER18D-145] WP_047824912.1 MULTISPECIES: aldolase [Massilia] KPK20478.1 hypothetical protein AMJ67_01080 [Betaproteobacteria bacterium SG8_41] WP_027197771.1 aldolase [Paraburkholderia sprentiae] SDR61564.1 Dihydrodipicolinate synthase/N-acetylneuraminate lyase [Paraburkholderia tuberum] WP_090812328.1 aldolase [Paraburkholderia tuberum] WP_077079464.1 MULTISPECIES: aldolase [Mycobacterium] WP_090422646.1 aldolase [Mycobacterium europaeum] WP_062895341.1 aldolase [Mycobacterium avium] WP_011856608.1 MULTISPECIES: aldolase [Mycobacteriaceae] WP_123787007.1 aldolase [Achromobacter denitrificans] WP_083173134.1 aldolase [Mycobacterium paraseoulense] WP_071394168.1 hypothetical protein [Bacillus tuaregi] WP_083094487.1 aldolase [Mycobacterium mantenii] ETZ38018.1 trans-2′-carboxybenzalpyruvate hydratase-aldolase [Mycobacterium intracellulare MIN_061107_1834] WP_009953931.1 MULTISPECIES: aldolase [Mycobacterium] WP_085290658.1 aldolase [Mycolicibacterium vulneris] RLA50226.1 aldolase [Gammaproteobacteria bacterium] WP_107764147.1 dihydrodipicolinate synthetase [Coprothermobacter proteolyticus] WP_007179239.1 aldolase [Burkholderia sp. Ch1-1] WP_067464354.1 aldolase [Actinomadura macra] WP_083829069.1 aldolase [Delftia sp. Cs1-4] AEF88778.1 dihydrodipicolinate synthetase [Delftia sp. Cs1-4] WP_086911711.1 aldolase [Acidovorax carolinensis] WP_036562639.1 aldolase [Oceanicola sp. MCTG156(1a)] TAD90455.1 aldolase [Alphaproteobacteria bacterium] WP_047824930.1 MULTISPECIES: aldolase [Massilia] WP_018963718.1 hypothetical protein [Coprothermobacter platensis] OGB50545.1 aldolase [Burkholderiales bacterium RIFOXYD12_FULL_59_19] GBD13407.1 Trans-2′-carboxybenzalpyruvate hydratase-aldolase [bacterium HR24] WP_066198397.1 aldolase [Hydrogenibacillus schlegelii] WP_007298126.1 MULTISPECIES: aldolase [Rhodococcus] WP_051423516.1 hypothetical protein [Arthrobacter sp. MA-N2] WP_117329621.1 dihydrodipicolinate synthetase [Sphaerochaeta halotolerans] WP_128644286.1 dihydrodipicolinate synthetase [Rhodococcus opacus] WP_087561951.1 MULTISPECIES: dihydrodipicolinate synthetase [Rhodococcus] WP_012642744.1 aldolase [Thermomicrobium roseum] WP_017681823.1 MULTISPECIES: aldolase [Rhodococcus] WP_124259333.1 aldolase [Rhodococcus ruber] TAN29949.1 hypothetical protein EPN30_01545 [Actinobacteria bacterium] WP_005570095.1 MULTISPECIES: aldolase [Rhodococcus] WP_005253631.1 aldolase [Rhodococcus opacus] WP_079931448.1 hypothetical protein [Gordonia sp. i37] AAR05109.1 putative aldolase [Rhodococcus sp. P400] OUS22376.1 hypothetical protein A9Q95_05145 [Rhodobacterales bacterium 59_46_T64] WP_013602975.1 aldolase [Pseudarthrobacter phenanthrenivorans] GBD18589.1 Trans-O-hydroxybenzylidenepyruvate hydratase-aldolase [bacterium HR27] WP_110795628.1 aldolase [Rhodobacteraceae bacterium FSX-11] WP_013255920.1 dihydrodipicolinate synthetase [Sediminispirochaeta smaragdinae] WP_075849231.1 aldolase [Saccharomonospora sp. CUA-673] WP_020501058.1 aldolase [Sciscionella marina] OUS03890.1 hypothetical protein A9Q96_17015 [Rhodobacterales bacterium 52_120_T64] WP_091675950.1 MULTISPECIES: aldolase [Amycolatopsis] WP_038532000.1 aldolase [Amycolatopsis methanolica] WP_087059681.1 aldolase [Actinomycetales bacterium JB111] WP_092817818.1 hypothetical protein [Halopenitus malekzadehii] WP_065123170.1 aldolase [Mycobacterium asiaticum] WP_107447362.1 aldolase [Streptomyces sp. P3] WP_067937422.1 aldolase [Mycobacterium sp. E2479] WP_027943869.1 aldolase [Amycolatopsis taiwanensis] WP_078947647.1 aldolase [Streptomyces griseus] WP_121792642.1 aldolase [Aeromicrobium sp. 9W16Y-2] WP_010204520.1 aldolase [Salinibacterium sp. PAMC 21357] AMK37583.1 trans-o-hydroxybenzylidenepyruvate hydratase-aldolase [Pseudomonas sp. C5pp] WP_087622569.1 aldolase [Aeromicrobium sp. PE09-221] WP_032395674.1 MULTISPECIES: aldolase [Rhodococcus] WP_039615401.1 MULTISPECIES: aldolase [Pseudomonas]

Cloning, and expression: DNA encoding heterologous aldolase hydratase enzymes were codon-optimized for expression in E. coli and synthesized by a commercial DNA synthesis company. Using standard cloning methods, each gene was cloned downstream of the T7 RNA polymerase promoter and upstream of the T7 terminator sequence in pB11 backbone plasmid. Additionally, for experiments wherein the aldehyde selected was 3-hydroxy-propionaldehyde a glycerol dehydratase enzyme that is a B12-dependent enzyme (Lactococcus reuteri glycerol dehydratase that is comprised of five genes as follows: pduC [Uniprot ID No. A5VMB2]; pduD [Uniprot ID No. A5VMB1]; pduE [Uniprot ID No. A5VMB0]; pduG [Uniprot ID No. A5VMA9]; and pduH [Uniprot ID No. A5VMA8]) was also cloned on a second compatible plasmid to enable production of 3-hydroxy-propionaldehyde from glycerol using this enzyme. The plasmids were transformed in E. coli BL21*(DE3) ΔldhA. Starter cultures for each clone were grown overnight in tubes containing 5 mL 2×YT media with 1 g/L D-glucose and appropriate antibiotics. Cell cultures for expression were carried out in 2 mL growth medium in 96 well plates. Complex (2×YT) growth medium was used and supplemented with 2 g/L D-glucose, 0.5 g/L potassium phosphate buffer (pH 7.2), and 100 mg/L ferric ammonium citrate. Pre-induction growth was carried out for 2 hours under aerobic conditions and at 30° C. Recombinant protein expression was induced at an OD600 of 0.2-0.4 with 250 μM IPTG. Post-induction expression was carried out for 30-180 minutes at 30° C. and under aerobic conditions followed by 0-60 mins under anaerobic conditions.

Enzyme assay: Post expression, cells were harvested and re-suspended in 0.4 mL fresh medium (0D600˜30) containing 15 g/L potassium phosphate buffer (pH 7.2) with substrates for the reaction. For activity determination, pyruvate (10-20 g/L) was incubated with 5-40 g/L aldehydes (e.g., acetaldehyde, propionaldehyde, butyraldehyde, 2-hydroxy-acetaldehyde, or 4-hydroxy-butyraldehyde) for 12 hr aerobically. For activity determination with 3-hydroxy-propanal, post expression cells were harvested and re-suspended in 0.4 mL fresh medium (0D600˜30) containing 15 g/L potassium phosphate buffer (pH 7.2) with 10-20 g/L glucose, 5-10 g/L glycerol, and 10 g/L pyruvate for 15 hr under anaerobic conditions. The reaction mix was also supplemented with 10 μM vitamin B12 and 1 g/L glutathione. After incubation at room temperature, the cells were centrifuged, and the supernatant was filtered and analyzed via HPLC.

Analysis of product: Isocratic HPLC was primarily used to detect and quantify production of enzyme products, aldol addition products (4-hydroxy-2-keto-carboxylic acids), aldol condensation products (3,4-dehydro-2-keto-carboxylic acids). One method employed a Bio-Rad Aminex HPX-87 column, 0.7 mL/min of 0.05% formic acid (or 5 mM sulfuric acid) at 35° C. Detection was carried out using an RID (refractive index detector) and UV detector, the latter of which was used to measure signals at 210 and 260 nm. Additionally, aldol addition and aldol condensation products were also confirmed by LC-MS, by measuring the masses of the respective peaks identified previously via HPLC (data not included herein).

Example 2: Enzymes that Catalyze Reduction of Aldol-Dehydration Products

As demonstrated herein, reduction of activated double bonds, i.e., double bonds next to a carbonyl or carboxylate group, can be catalyzed by enzymes. Aldol-dehydration products, e.g., 2-oxo-3-enoic acids, can be further reduced using enzymes, to give the corresponding 2-oxo-carboxylic acids. It was unexpectedly discovered that oxidoreductases belonging to EC 1.6.5 (e.g., EC 1.6.5.5) that utilize NADH and/or NADPH for reduction of quinones are capable of catalyzing this reaction. For example, when Ads-Hyd enzymes (see Example 1) were recombinantly expressed in E. coli BL21 or E. coli MG1655 strains for the production of 2-keto-carboxylic acids as described in Example 1, it was discovered that a portion of the Ads-Hyd enzyme product (i.e., 2-oxo-3-enoic acids) was converted to the corresponding 2-keto-carboxylic acid. This led to the possibility that some natively expressed enzyme or enzymes within these E. coli strains was responsible for carrying out the reduction of 2-oxo-3-enoic acids. A survey of known oxidoreductases that could conceivably carry out reduction of activated double bonds (i.e., EC 1.3.- and EC 1.6.-) within these strains was carried out. Seventeen such promising enzymes were identified within E. coli MG1655 and E. coli BL21 each. Knock-out strains for each of these enzymes in both of these hosts were prepared using known methods in the art. Subsequently each such knockout strain was tested for its ability to produce both of 2-oxo-3-enoic acid and its product of 2-keto-carboxylic acid using methods described above and using recombinantly expressed Ads-Hyd enzymes. This led to identification that knocking out the qorA gene or quinone oxidoreductase-1 led to production of 2-oxo-3-enoic acid and no 2-keto-carboxylic acid. This confirmed that the enzyme encoded by the qorA was likely responsible for natively carrying out this reaction. Subsequently, a N-terminal His6 tagged QorA enzyme (“His6” disclosed as SEQ ID NO: 106) was overexpressed and purified, and it was confirmed that it was indeed active for carrying out the desired reaction (FIG. 6). This unequivocally confirmed for the first time that quinone oxidoreductase enzyme from E. coli belonging to EC 1.6.5 (e.g., EC 1.6.5.5) is capable of functioning on substrates that are very different from their natural substrates, which are cyclic in structure. Furthermore, it was confirmed that this enzyme is able to utilize both NADH and NADPH as cofactors during the reaction (FIG. 6), which is very advantageous as it enables use of this enzyme under both aerobic and anaerobic conditions during bioproduction.

Various biosynthesis polypeptides belonging to EC 1.6.5 can be utilized in accordance with the present disclosure, e.g., as alkene reduction product biosynthesis polypeptides and/or for reduction of aldol-dehydration products. For example, a number of quinone oxidoreductases of EC 1.6.5.5 were assessed for their activities in accordance with the present disclosure, including eighteen enzymes (see Table 9) whose identities to E. coli Qor-1 enzyme ranged from 37-90%. All enzymes selected were confirmed to be active on at least one substrate (Table 9), further confirming the generality of this class of enzymes to carry out this reaction.

TABLE 9 Certain useful biosynthesis polypeptides - reductase. Activity on Different substrates 6-hydroxy-3,4- Enzyme Enzyme % Identity Uniprot ID or 3,4-dehydro-2- 3,4-dehydro-2- dehydro-2- Name ID to Qor-1 Genbank ID oxopentanoate oxohexanoate oxohexanoate Ec QorA Qor-1 100 P28304 + + + Stm Qor Qor-2 90 P40783 NT NT + Reh Qor1 Qor-3 43 Q0K2I0 NT NT + Pvl Qor Qor-4 67 A0A1Z1SRY9 NT NT + Pae Qor Qor-5 59 P43903 NT NT + Msg Qor Qor-6 44 I7G8G0 NT NT + Bxb Qor Qor-7 48 Q142L2 NT NT + Bcep Qor Qor-8 48 ALK19324.1 NT NT + Aalbi Qor Qor-9 42 A0A1G9R408 NT NT + Ain Qor Qor-10 29 G4Q8R5 NT NT + Mche Qor Qor-11 37 ANA98723.1 NT NT + Nbr Qor Qor-12 42 K0EUQ3 NT NT + Pole Qor Qor-13 60 A0A061CRS8 NT NT + Ccr Qor Qor-14 46 Q9A212 NT NT + Sflav Qor Qor-15 42 A0A1I6RWW2 NT NT + Smari Qor Qor-16 44 WP_026197277.1 NT NT + Zmo Qor Qor-17 37 Q5NKZ3 NT NT + Met Qor Qor-18 48 WP_012333034.1 NT NT + Tri Qor Qor-19 47 WP_136898000.1 NT NT + NT = Not tested; NA = Not active; + = activity confirmed but not quantified Other reduction product biosynthesis polypeptides, e.g., those belonging to various subclasses of EC 1.6.5 such as various quinone oxidoreductase enzymes belonging to EC 1.6.5.5 may also carry out this reaction.

Cloning and expression: DNA encoding heterologous aldolase hydratase (Ads-Hyd 1) and quinone oxidoreductase enzymes shown in Table 5 were codon-optimized for expression in E. coli and synthesized by a commercial DNA synthesis company. For in vitro activity measurements, and N-terminal His6 tag (SEQ ID NO: 106) was added onto Qor-1 enzyme. Using standard cloning methods, each gene was cloned downstream of the T7 RNA polymerase promoter and upstream of the T7 terminator sequence in single pB11 backbone plasmid. Additionally, for experiments wherein the aldehyde selected was 3-hydroxy-propionaldehyde a glycerol dehydratase enzyme that is a B12-dependent enzyme (Lactococcus reuteri glycerol dehydratase that is comprised of five genes as follows: pduC [Uniprot ID No. A5VMB2]; pduD [Uniprot ID No. A5VMB1]; pduE [Uniprot ID No. A5VMB0]; pduG [Uniprot ID No. A5VMA9]; and pduH [Uniprot ID No. A5VMA8]) was also cloned on a second compatible plasmid to enable production of 3-hydroxy-propionaldehyde from glycerol using this enzyme. The plasmids were transformed in E. coli BL21*(DE3) ΔldhA ΔqorA. Recombinant protein expression was carried out as described above in Example 1. For in vitro studies, the Qor-1 enzyme was induced at an OD600 of 0.2-0.4 with 250 μM IPTG. Post-induction expression was carried out for 180 minutes at 30° C. and under aerobic conditions. Post induction the enzyme was purified using Ni-NTA affinity chromatography using standard methods in art.

Enzyme assay: Same as Example 1 of in vivo activity measurement of the different quinone oxidoreductases. For in vitro activity measurement shown FIG. 6, the Qor-1 enzyme (0.3 mg/ml) was incubated with ˜10 mM of 6-hydroxy-3,4-dehydro-2-oxohexanoate (synthesized in house), 0.5 mM of either NADH or NADPH in 100 mM pH 7 phosphate buffer.

Analysis of product: Isocratic HPLC method described in Example 1 was used to detect and quantify production of enzyme product, i.e., 2-keto-carboxylic acids. For in vitro activity measurement, the decrease in absorbance at 340 nm was used to measure depletion of NADH or NADPH cofactor and thus Qor-1 activity.

Example 3: A Two-Enzyme System for the Production of 2-Keto-Carboxylic Acids from Pyruvate and Aliphatic Aldehydes

The use of aldolase-hydratase enzyme(s) in combination with quinone oxidoreductase enzymes for the production of a range of 2-keto acids was examined. This combination enables the production of a range of 2-keto acids, which are precursors for the production of a number of industrially desirable products such as 1,5-pentanediol, 1,6-hexanediol, adipic acid, caprolactam, caprolactone, 6-hydroxy hexanoic acid, 6-amino caproic acid, amino acids, and many different fatty molecules. A number of different combinations of aldolase-hydratase enzymes and oxidoreductases were confirmed to be active for the production of different 2-keto acids (Table 10). As demonstrated herein, provided technologies can provide high products concentration, e.g., about or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 15, 17, 18, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1500, 2000, 2500, 3000 mM.

TABLE 10 Provided technologies comprising multiple biosynthesis polypeptides generate desired products. Activity on Different substrates mM of mM of mM of Reduc- 2-keto 2-keto 6-hydroxy- tase pentanoic hexanoic 2-keto hexanoic Ads-Hyd ID ID acid product acid product acid product Ads-Hyd 1 Qor-1 + + 3.2 Ads-Hyd 2 Qor-1 NT NT + Ads-Hyd 3 Qor-1 NT NT NA Ads-Hyd 4 Qor-1 NT NT 7.1 Ads-Hyd 5 Qor-1 NT NT NA Ads-Hyd 6 Qor-1 NT NT + Ads-Hyd 7 Qor-1 NT NT + Ads-Hyd 8 Qor-1 NT NT 5.8 Ads-Hyd 9 Qor-1 NT NT + Ads-Hyd 10 Qor-1 NT NT 12.3 Ads-Hyd 11 Qor-1 NT NT + Ads-Hyd 12 Qor-1 NT NT + Ads-Hyd 13 Qor-1 NT NT + Ads-Hyd 14 Qor-1 NT NT + Ads-Hyd 15 Qor-1 NT NT + Ads-Hyd 62 Qor-1 NT NT 20.0 Ads-Hyd 87 Qor-1 NT NT 28.4 Ads-Hyd 96 Qor-1 NT NT 28.3 Ads-Hyd 104 Qor-1 NT NT 24.6 Ads-Hyd 65 Qor-1 NT NT 18.9 Ads-Hyd 89 Qor-1 NT NT 8.5 Ads-Hyd 97 Qor-1 NT NT 26.1 Ads-Hyd 68 Qor-1 NT NT 18.5 Ads-Hyd 108 Qor-1 NT NT 33.8 Ads-Hyd 29 Qor-1 NT NT 18.3 Ads-Hyd 69 Qor-1 NT NT 8.9 Ads-Hyd 93 Qor-1 NT NT 40.5 Ads-Hyd 8 Qor-1 NT NT 5.8 Ads-Hyd 8 Qor-2 NT NT + Ads-Hyd 8 Qor-3 NT NT + Ads-Hyd 8 Qor-4 NT NT + Ads-Hyd 8 Qor-5 NT NT + Ads-Hyd 8 Qor-6 NT NT + Ads-Hyd 8 Qor-7 NT NT + Ads-Hyd 8 Qor-8 NT NT + Ads-Hyd 8 Qor-9 NT NT + Ads-Hyd 8 Qor-10 NT NT + Ads-Hyd 8 Qor-11 NT NT + Ads-Hyd 8 Qor-12 NT NT + Ads-Hyd 8 Qor-13 NT NT + Ads-Hyd 8 Qor-14 NT NT + Ads-Hyd 8 Qor-15 NT NT + Ads-Hyd 8 Qor-16 NT NT + Ads-Hyd 8 Qor-17 NT NT + Ads-Hyd 8 Qor-18 NT NT + Ads-Hyd 8 Qor-19 NT NT + NT = Not tested; NA = Not active; + = activity confirmed but not quantified

Various biosynthesis polypeptides, particularly those belonging to EC 1.6.5, may be utilized for a reduction. For example, quinone oxidoreductases belonging to EC 1.6.5.5. are reported to be involved in electron carrier activity and are reported to be ubiquitous enzymes as they are reported to be present in, e.g., mammals, fungi, and bacteria (see entry for this EC class on Brenda.org). Although the native expression levels of these enzymes across various hosts are not known, it has been postulated previously that the expression level of this class of enzymes natively can be affected by the oxidative stress faced by the microbial host. It was discovered that E. coli (MG1655 and BL 21 strains) QorA gene (Qor-1) is natively expressed, especially under conditions described in Example 2. It was demonstrated that even native enzyme levels of Qor-1 in E. coli can be sufficient for production of 2-keto acids when Ads-Hyd enzymes (e.g., Ads-Hyd 8) are overexpressed in E. coli. For example, when Ads-Hyd 8 is overexpressed in E. coli BL 21*(DE3) ΔldhA, this resulted in the production of ˜3 mM 6-hydroxy 2-keto hexanoate. However, overexpression of Qor-1 from plasmids in addition to Ads-Hyd 8, led to ˜2× improved production (˜5.8 mM 6-hydroxy 2-keto hexanoate). Based on this result, the in vitro kinetics data gathered in-house, and typical enzyme levels discovered in E. coli, it is estimated that in some embodiments, the native amounts of Qor-1 enzyme expressed under these conditions is <100 μM, and likely in the range of 0.1-100 μM.

Compared to a three-enzyme system, wherein aldol addition, dehydration, and subsequent reduction are carried out by three separate enzymes, provided technologies using two-enzyme systems provided significant improvement, for example: (1) only two enzymes need to be expressed rather than three enzymes—thus reducing catalysts required, and reducing cell resources for protein production when reaction are conducted in vivo, and (2) by having a single biosynthesis polypeptide carry out both the aldol addition and condensation reactions, reaction equilibrium is shifted towards the direction of production of desired products, which can be favorable to overall yields feasible through the process.

Cloning, and expression: DNA encoding heterologous aldolase hydratases and quinone oxidoreductase enzymes shown in Table 5 were codon-optimized for expression in E. coli and synthesized by a commercial DNA synthesis company. Using standard cloning methods, each gene was cloned downstream of the T7 RNA polymerase promoter and upstream of the T7 terminator sequence on two compatible plasmids. Additionally, for experiments wherein the aldehyde selected was 3-hydroxy-propionaldehyde, a glycerol dehydratase enzyme that is a B12-dependent enzyme (Lactococcus reuteri glycerol dehydratase that is comprised of five genes as follows: pduC [Uniprot ID No. A5VMB2]; pduD [Uniprot ID No. A5VMB1]; pduE [Uniprot ID No. A5VMB0]; pduG [Uniprot ID No. A5VMA9]; and pduH [Uniprot ID No. A5VMA8]) was also cloned on a third compatible plasmid to enable production of 3-hydroxy-propionaldehyde from glycerol using this enzyme. The plasmids were transformed in E. coli MG1655 (DE3) rne131 ΔldhA ΔadhE ΔfrdBC ΔpoxB ΔpflB ΔackA-pta ΔyqhD, ΔadhP, ΔeutG, ΔgldA, ΔyiaY, ΔfucO. Recombinant protein expression was carried out as described above in Example 1.

Enzyme assay: Same as Example 1.

Analysis of product: Isocratic HPLC method described in Example 1 was used to detect and quantify production of enzyme product, i.e., 2-keto-carboxylic acids.

Example 4: Biosynthetic Pathway for the Production of 1,5-Pentanediol

This example describes a biosynthetic pathway for the production of 1,5-pentanediol from pyruvate and 3-hydroxy-propionaldehyde. As shown in FIG. 2, the biosynthetic pathway from pyruvate and 3-hydroxy-propionaldehyde includes five reactions. The first three reactions are described in Example 3, which involve converting pyruvate and 3-hydroxy-propionaldehyde to 6-hydroxy-2-keto-hexanoate. Described below are both known enzymes from the remaining two steps of the pathway. Notably, enzymes have been validated for all five reactions, which included demonstrating the complete pathway in vivo (see Example 5).

Steps 1-3: Conversion of pyruvate and 3-hydroxy-propionaldehyde to 6-hydroxy-2-oxo-hexanoate. See Example 3 for details.

Step 4: Conversion of 6-hydroxy-2-oxo-hexanoate to 5-hydroxy-pentanal. Exemplary enzymes are shown in Table 11. 2-Keto-acid decarboxylases (EC 4.1.1.7) catalyze the thiamine diphosphate (TPP) dependent decarboxylation of (CO 2-keto acids to give the corresponding (C_(n-1)) aldehydes. Enzymes that possess high-activity towards long-chain 2-oxo-acids with minimal or no activity on pyruvate are desired since cross-reactivity with pyruvate can dramatically affect yields of this pathway. Z. mobilis pyruvate decarboxylase (PDC) has been mutated (I472A/I476F) to significantly modify its active site for increased efficiency towards long-chain 2-oxo-acids along with a dramatic reduction (>2000 fold) in its activity towards pyruvate.⁷ Z. mobilis PDC mutant 1472A/I476F also shows excellent kinetic properties on 2-oxo-hexanoate which is structurally similar to desired substrate. Another promising enzyme candidate for catalyzing this step is L. lactis branched chain keto-acid decarboxylase KdcA (ketoacid decarboxylase), and P. putida benzoyl formate decarboxylase (BFD) mutant A460I.⁸⁻¹⁰ The Pseudomonas putida BFD and L. lactis KdcA show >50 and 500-fold selectivity towards long-chain 2-oxo-acids compared to pyruvate for decarboxylation. In particular, L. lactis KdcA has specific activity towards 2-oxo-hexanoic acid and can tolerate substitutions on C3 and C4 positions. This enzyme was confirmed to be active for catalyzing the decarboxylation reaction (Table 14).

TABLE 11 Exemplary enzymes. Uniprot Gene E.C. ID Protein Name Name Organism Number Q6QBS4 Branched-chain alpha- kdcA Lactococcus 4.1.1.72 ketoacid decarboxylase lactis A7M7D6 Pyruvate decarboxylase pdc Zymomonas 4.1.1.1 mobilis P20906 benzoyl formate mdlc Pseudomonas 4.1.1.7 decarboxylase putida

Decarboxylases having other EC numbers are also suitable for carrying out this reaction. A representative list is shown in Table 12.

TABLE 12 Exemplary decarboxylases. E.C. Number Name 4.1.1.1 Pyruvate decarboxylase 4.1.1.2 Oxalate decarboxylase 4.1.1.3 oxaloacetate decarboxylase 4.1.1.4 acetoacetate decarboxylase 4.1.1.5 acetolactate decarboxylase 4.1.1.6 aconitate decarboxylase 4.1.1.7 benzyl formate decarboxylase 4.1.1.11 aspartate-1-decarboxylase 4.1.1.12 aspartate-4-decarboxylase 4.1.1.15 glutamate decarboxylase 4..1.1.16 hydroxyglutamate decarboxylase 4.1.1.17 ornithine decaraboxylase 4.1.1.18 lysine decarboxylase 4.1.1.19 arginine decarboxylase 4.1.1.20 diaminopimelate decarboxylase 4.1.1.34 dehydro-L-gulonate decarboxylase 4.1.1.35 UDP-glucuronate decarboxylase 4.1.1.40 hydroxypyruvate decarboxylase 4.1.1.54 dihydroxyfumarate decarboxylase 4.1.1.56 3-oxolaurate decarboxylase 4.1.1.71 2-oxoglutarate decarboxylase 4.1.1.72 branched chain 2-oxo-acid decarboxylase 4.1.1.73 tartarate decarboxylase 4.1.1.74 indolepyruvate decarboxylase 4.1.1.75 5-guanidino-2-oxopentanoate decarboxylase 4.1.1.77 2-oxo-3-hexnedioate decarboxylase

Step 5: Conversion of 5-hydroxy-pentaldehyde to 1,5-pentanediol. Primary alcohol dehydrogenases catalyze the NAD(P)H-dependent reduction of aldehydes to primary alcohols.

Many primary alcohol dehydrogenases are known in literature, and exemplary candidates to catalyze this step are described below and shown in Table 13 below. A number of E. coli alcohol-aldehyde dehydrogenases are known including AdhE, adhP, eutG, yiaY, yqhD, fucO, and yjgB.¹¹ Recently, 44 aldehyde reductases have been identified in E. coli. Butanol dehydrogenases¹² from C. acetobutylicum are of interest to catalyze these transformations. A number of S. cerevisiae alcohol dehydrogenases have been shown to reduce a range of different aldehydes including, ADH2-6. Of particular interest is ADHI-ADHII from two alkyl alcohol dehydrogenase (ADH) genes¹³ from the long-chain alkane-degrading strain Geobacillus thermodenitrificans NG80-2. Other promiscuous ADH include AlrA which encodes a medium-chain alcohol dehydrogenase.¹⁴ Also of interest are 4-hydroxy butyrate dehydrogenases (EC 1.1.1.61) that catalyze reduction of 4-oxo butyrate that have been found in A. thaliana ¹⁵ , E. coli (yihu)¹⁶, and as well as C. Eluyveri.¹⁷ A. thaliana enzyme as well as A. terrus enzyme (ATEG in Table 13) can reduce glutarate semialdehyde (WO 20101068953A2, WO 20101068953A2). Although a number of alcohol dehydrogenase are of interest for carrying out this reaction, a specific enzyme of particular interest due to its high level of activity for reducing 5-hydroxy pentanal is alcohol dehydrogenase from Leifsonia sp. S749 (GenBank ID No. AB213459.1). This enzyme and four other alcohol dehydrogenases were validated (Table 14) to carry out this reaction.

TABLE 13 Exemplary dehydrogenases. GenBank ID or Gene Uniprot ID Name Organism fucO NP_417279.1 Alcohol Dehydrogenase Escherichia coli bdh I NP_349892.1 Alcohol Dehydrogenase Clostridium acetobutylicum bdh II NP_349891.1 Alcohol Dehydrogenase Clostridium acetobutylicum alrA BAB12273.1 Alcohol Dehydrogenase Acinetobacter sp. strain 4hbd L21902.1 4-hydroxy butyrate Clostridium kluyveri dehydorgenase 4hbd Q94B07 4-hydroxy butyrate Arabidopsis thaliana dehydorgenase yihu AAB03015.1. 4-hydroxy butyrate Escherichia coli dehydorgenase ADH2 NP_014032.1 Alcohol Dehydrogenase Saccharomyces cerevisiae ADH3 NP_013892.1 Alcohol Dehydrogenase Saccharomyces cerevisiae ADH4 NP_015019.1 Alcohol Dehydrogenase Saccharomyces cerevisiae ADH5 NP_010996.2 Alcohol Dehydrogenase Saccharomyces cerevisiae ADH6 ABX39192.1 Alcohol Dehydrogenase Saccharomyces cerevisiae ATEG XP_001210625.1 Alcohol Dehydrogenase Aspergillus terreus ADHI ABO67118 Alcohol Dehydrogenase Geobacillus thermodenitrificans NG80-2 ADHII ABO68223 Alcohol Dehydrogenase Geobacillus thermodenitrificans NG80-2 YqhD BAE77068.1 Alcohol Dehydrogenase Escherichia coli bdh CLJU_c23460 D8GL45 butanol dehydrogenase Clostridium ljungdahlii bdhA CA_C3299 Q04944 butanol dehydrogenase A Clostridium acetobutylicum chnD Q84H78 6-hydroxyhexanoate Rhodococcus sp. Phi2 dehydrogenase chnD Q7WVD0 6-hydroxyhexanoate Acinetobacter sp. NCIMB9871 dehydrogenase lsadh AB213459.1 Short chain alcohol Leifsonia sp. S749 dehydrogenase Adhe CAA47743.1. Alcohol Dehydrogenase Escherichia coli

Cloning, and expression: DNA encoding heterologous 2-keto acid decarboxylase and alcohol dehydrogenase enzymes shown in Table 14 below were codon-optimized for expression in E. coli and synthesized. Using standard cloning methods, each gene was cloned downstream of the T7 RNA polymerase promoter and upstream of the T7 terminator sequence on a single plasmid. The plasmid was transformed in E. coli MG1655 (DE3) rne131 ΔldhA ΔadhE ΔfrdBC. Recombinant protein expression was carried out as described above in Example 1.

TABLE 14 Production of 1,5-pentanediol. Uniprot ID of 1,5-pentanediol Example Uniprot ID of Keto primary alcohol produced No: acid decarboxylase dehydrogenase* (g/L) 4A Q6QBS4 D8GL45 0.6 4B Q6QBS4 Q04944 0.8 4C Q6QBS4 Q84H78 1.4 4D Q6QBS4 Q7WVD0 1.4 4E Q6QBS4 AB213459.1 1.4 *In this case, this enzyme also can be referred to as 5-hydroxy-pentanal 1-reductase.

Activity Assay: Observation of the production of 1,5-pentanediol from externally fed 6-hydroxy-2-keto-hexanoate indicated successful activity of the 2-keto acid decarboxylase and alcohol dehydrogenase enzymes. Thus post expression, cells were harvested and re-suspended in 0.4 mL fresh medium (0D600-30) containing 15 g/L potassium phosphate buffer (pH 7.2) with 6-hydroxy-2-keto-hexanoate (˜5 g/L) and 10 g/L glucose, for 15 hr under anaerobic conditions. After incubation at room temperature, the cells were centrifuged, and the supernatant was filtered and analyzed via HPLC for the formation of 1,5-pentanediol from 6-hydroxy-2-keto-hexanoate.

HPLC analysis of 1,5-pentanediol production: Isocratic HPLC was used to detect and quantify 1,5-pentanediol. The method employed a Bio-Rad Aminex HPX-87 column, 0.7 mL/min of 0.05% formic acid (or 5 mM sulfuric acid) at 35° C. Detection was carried out using an RID (refractive index detector) and UV detector, the latter of which was used to measure signals at 210 and 260 nm. The HPLC results showed production of 1,5-pentanediol; results of certain preparations were presented in Table 14.

Example 5: Preparation and Use of Microbial Organism for Production of 1,5-Pentanediol from Different Carbon Sources Via 6-hydroxy-2-keto-hexanoate Intermediate

In some embodiments, the present disclosure provides technologies for producing 1,5-pentanediol. In some embodiments, glycerol is utilized as a carbon source. In some embodiments, one or more, or all, biosynthesis steps are performed in one organism (e.g., bacterium) and culture. In some embodiments, a yield is about or at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 mg/L, or is about or at least about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.7, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 250, or 300 g/L.

E. coli was used as an exemplary organism to engineer the production of 1,5-pentanediol from carbon sources such as glycerol and/or glucose via metabolic precursor pyruvate and 3-hydroxy-propionaldehyde that are derived from these carbon sources, using the metabolic pathway which is shown in FIG. 2, and which is also described in Example 4. To generate E. coli capable of making 1,5-pentanediol via this pathway from desired carbon sources (e.g. glycerol and/or glucose), the nucleic acid encoding each individual enzyme in the pathway and other enzymes necessary for 3-hydroxy-propionaldehyde production were either codon-optimized for E. coli and synthesized commercially or obtained via PCR amplification using E. coli genomic DNA. Genes were cloned into plasmids, which were transformed in E. coli. In vivo expression of all of the pathway enzymes resulted in production of 1,5-pentanediol.

Cloning of 1,5-pentanediol pathway genes: DNA encoding heterologous enzymes in the 1,5-pentanediol pathway were codon-optimized for expression in E. coli and synthesized by a commercial DNA synthesis company (e.g., Twist Biosciences). DNA encoding native enzymes in the 1,5-pentanediol pathway were amplified from E. coli genomic DNA via PCR. Using standard cloning methods, each gene was cloned downstream of the T7 RNA polymerase promoter and upstream of a terminator sequence. Compatible plasmids harboring expression cassettes for the genes contained one of the following combinations of a marker and replicon: (1) chloramphenicol maker+P15A replicon, (2) ampicillin marker+ColE1 replicon, and (3) kanamycin marker+COLA replicon. Examples of genes used include the following: Ads-Hyd 8 (Uniprot ID No. A0A286PH18), Qor-1 (Uniprot ID No. P28304), 6-hydroxy-2-oxo-hexanoate decarboxylase (Uniprot ID No. Q6QBS4), primary alcohol dehydrogenase also referred to as 5-hydroxy-pentanal 1-reductase (GenBank ID No. AB213459.1). Additionally, glycerol dehydratase enzyme that is vitamin B12-independent (e.g. Clostridium butyricum glycerol dehydratase that is comprised of two subunits as follows: DhaB1 [Uniprot ID No. Q8GEZ8]; DhaB2 [Uniprot ID No. Q8GEZ7]) or glycerol dehydratase enzyme that is a B12-dependent enzyme (Lactococcus reuteri glycerol dehydratase that is comprised of five genes as follows: pduC [Uniprot ID No. A5VMB2]; pduD [Uniprot ID No. A5VMB1]; pduE [Uniprot ID No. A5VMB0]; pduG [Uniprot ID No. A5VMA9]; and pduH [Uniprot ID No. A5VMA8]) was also cloned to enable production of 3-hydroxy-propionaldehyde—a 1,5-pentanediol pathway precursor that can be made from glycerol using this enzyme. All five genes encoding the Lactococcus reuteri glycerol dehydratase were cloned as a single gene operon.

Construction of strain(s) for the production of 1,5-pentanediol: The E. coli strain BL21* (DE3) ΔldhA was used as the background strain for testing of the 1,5-pentanediol pathway enzymes. Plasmids harboring the genes encoding the pathway enzymes were transformed using standard electroporation methods associated with transforming E. coli.

Production of 1,5-pentanediol: The following expression strains were obtained after sequentially transforming the following plasmids into E. coli.

Strain PeDO1: Plasmid 1 (COLA replicon, kanamycin marker): Gene 1 (Glycerol dehydratase—DhaB1), Gene 2 (Glycerol dehydratase—DhaB2), Gene 3 (Qor 1). Plasmid 2 (ColE1 replicon, ampicillin marker): Gene 1 (6-hydroxy-2-oxo-hexanoate decarboxylase), Gene 2 (Ads-Hyd 8). Plasmid 3 (P15A replicon, chloramphenicol marker): Gene 1 (5-hydroxy-pentanal 1-reductase).

Strain PeDO2: Plasmid 1 (COLA replicon, kanamycin marker): Gene 1 (Glycerol dehydratase—DhaB1), Gene 2 (Glycerol dehydratase—DhaB2), Gene 3 (Qor 1). Plasmid 2 (ColE1 replicon, ampicillin marker): Gene 1 (6-hydroxy-2-oxo-hexanoate decarboxylase), Gene 2 (Ads-Hyd 8).

Strain PeDO3: Plasmid 1 (COLA replicon, kanamycin marker): Gene 1 (Glycerol dehydratase—pduCDEGH). Plasmid 2 (ColE1 replicon, ampicillin marker): Gene 1 (6-hydroxy-2-oxo-hexanoate decarboxylase), Gene 2 (Ads-Hyd 8), Gene 3 (5-hydroxy-pentanal 1-reductase). Plasmid 3 (P15A replicon, chloramphenicol marker): Gene 1 (Qor 1).

Strain PeDO4: Plasmid 1 (COLA replicon, kanamycin marker): Gene 1 (Glycerol dehydratase—pduCDEGH). Plasmid 2 (ColE1 replicon, ampicillin marker): Gene 1 (6-hydroxy-2-oxo-hexanoate decarboxylase), Gene 2 (Ads-Hyd 8). Plasmid 3 (P15A replicon, chloramphenicol marker): Gene 1 (5-hydroxy-pentanal 1-reductase).

Culturing for Strain PeDO1 and PeDO2: Starter cultures were grown overnight in tubes containing 5 mL 2×YT media with 1 g/L D-glucose and appropriate antibiotics. Cell cultures for the expression and the 1,5-pentanediol pathway enzymes were carried out in 40 mL growth medium using 125 mL baffled flasks. Complex (2×YT) growth medium was used and supplemented with 2 g/L D-glucose, 0.5 g/L potassium phosphate buffer (pH 7.2), and 100 mg/L ferric ammonium citrate. Pre-induction growth was carried out for 2 hours under aerobic conditions and at 30° C. Recombinant protein expression was induced at an OD600 of 0.2-0.4 with 250 μM IPTG. Post-induction expression was carried out for 30 minutes at 30° C. and under aerobic conditions. Cell cultures were then transferred to 100 mL glass bottles, L-cysteine-HCl-monohydrate was added to the growth medium (1 g/L final concentration), and the bottles were sealed within an anaerobic glove box (Coy Laboratory). Cultures were then grown in the glass bottles for 2 hours at 30° C. and under anaerobic conditions. Afterwards, cells were harvested and re-suspended in 0.4 mL fresh medium (0D600˜30) containing 8 g/L glucose, 4 g/L glycerol, and 15 g/L potassium phosphate buffer (pH 7.2). After incubation under anaerobic conditions for 24 hours and at room temperature, the cells were centrifuged, and the supernatant was filtered and analyzed via HPLC.

Culturing for Strain PeDO3 and PeDO4: Production medium contains following composition: 1×MOPS minimal medium, 5 g/L yeast extract, 10 g/L glycerol, 20 g/L glucose, and 10 uM of Cyanocobalamin (pH7.2). The 1×MOPS minimal medium is composed of 40 mM MOPS, 4 mM tricine, 0.01 mM FeSO₄, 9.5 mM NH₄Cl, 0.276 mM K2504, 0.5 μM CaCl₂, 0.525 mM MgCl₂, 50 mM NaCl, 2.92E⁻⁷ mM (NH4)2MoO4, 4.0E⁻⁵ mM H₃BO₃, 3.02E⁻⁶ mM CoCl₂, 9.62E⁻⁷ mM CuSO₄, 8.08E⁻⁶ mM MnCl₂, 9.74E⁻⁷ mM ZnSO4, and 1.32 mM K₂PO₄. Seed cultures were grown overnight in tubes containing 10 mL 2×YT media and appropriate antibiotics. Cell cultures for 1,5-pentanediol production were prepared using 10 mL production medium with appropriate antibiotics in 125 mL flask with a stopper, 1 mL of seed culture was inoculated and allow cell to grow at 37° C. for 2 hr before induction. After 2 hr, cell culture was induced with 0.1 mM IPTG and the culture was transferred to 26° C. to start the production. Samples were taken every 12 hr aerobically with final sample taken at 72 hr, and the supernatant was filtered and analyzed via HPLC.

HPLC analysis of 1,5-pentanediol production: Isocratic HPLC was used to detect and quantify 1,5-pentanediol. The method employed a Bio-Rad Aminex HPX-87 column, 0.7 mL/min of 0.05% formic acid (or 5 mM sulfuric acid) at 35° C. Detection was carried out using an RID (refractive index detector) and UV detector, the latter of which was used to measure signals at 210 and 260 nm. The HPLC results showed evidence 1,5-pentanediol production at a final titer of 800 mg/L (Strain PeDO1), 400 mg/L (PeDO2), 212 mg/L (PeDO3), and 41 mg/L (PeDO4).

Additional Working Examples for 1,5-Pentanediol Production

Based on the success of producing 1,5-pentanediol using the above-described strains, the use of alternative quinone oxidoreductases identified in Examples 2 & 3 for the production of 1,5-pentanediol was assessed. Briefly, the plasmid combination of Strain PeDO3 in the above-described example was used, wherein the plasmid 3 contained different Qor enzymes namely Qor-1 (Uniprot ID No. P28304), Qor-2 (Uniprot ID No. P40783), and Qor-5 (Uniprot ID No. P43903). The strain construction, production, and analytical methods were identical to those described above. Strain PeDO5 (containing Qor-1), Strain PeDO6 (containing Qor-2), and Strain PeDO6 (containing Qor-5) led to the production of ˜2 g/L, 2.2 g/L and 2.4 g/L 1,5-pentanediol respectively under production conditions described above

Example 6: Preparation and Use of Microbial Organism for Production of 1,6-Hexanediol from 6-hydroxy-hexanoate Intermediate

In some embodiments, the present disclosure provides technologies for preparing 6HH and HDO. In some embodiments, a yield is about or at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 mg/L, or is about or at least about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.7, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 250, or 300 g/L. A biosynthetic pathway for the production of 1,6-hexanediol from 6-hydroxy-hexanoate (6HH) intermediate is shown in FIG. 3. Shown below are examples incorporating the use of different enzymes for each step of this pathway to validate the production of 1,6-hexanediol from 6HH. Examples of genes and corresponding enzymes from which they are encoded that were used to carry out each step of the 1,6-hexanediol biosynthetic pathway from 6HH intermediate are shown in Table 15 below. Each enzyme therein may be substituted with homologous enzymes that belong to the same E.C. class.

TABLE 15 Production of HDO. Gene 1 Gene 2 Gene 3 Uniprot ID or Uniprot ID or Uniprot ID or ENTRY No: Genbank ID Genbank ID Genbank ID 1 D6Z860 P39135 AB213459.1 2 YP_001705436.1 P39135 AB213459.1 3 ANO06407.1 P39135 AB213459.1 4 AAR91681.1 P39135 AB213459.1 5 AHH98121.1 P39135 AB213459.1 6 ANB00612.1 P39135 AB213459.1 7 ANO04655.1 P39135 AB213459.1 8 A0R484 P39135 AB213459.1 9 AFP42026.1 P39135 AB213459.1 10 GAJ86510.1 P39135 AB213459.1 11 YP_001704097.1 P39135 AB213459.1 12 ANA99315.1 P39135 AB213459.1 13 GAJ83027.1 P39135 AB213459.1 14 ANA98925.1 P39135 AB213459.1 15 ANA98924.1 P39135 AB213459.1 16 ANO04656.1 P39135 AB213459.1 17 YP_001703694.1 P39135 AB213459.1 18 WP_036338301.1 P39135 AB213459.1 19 WP_007472106.1 P39135 AB213459.1 20 A0QWI7 P39135 AB213459.1 Reaction catalyzed by enzyme named 6-hydroxyhexanoate 1-reductase, which is coded by gene 1: 6-hydroxy-hexanoate->6-hydroxy-hexanal. Enzyme coded by gene 2: 6-hydroxyhexanoate 1-reductase activator. Reaction catalyzed by enzyme named 6-hydroxyhexanal 1-reductase, which is coded by gene 3: 6-hydroxy-hexanal->1,6-hexanediol

(i) Preparation of Plasmids for HDO Production:

The HDO production pathway genes were cloned on a two plasmids shown below.

Synthetic genes were obtained from commercial vendors, and each gene was codon optimized for expression in E. coli. Each gene was cloned under its own T7 promoter and terminator using standard molecular biology methods. Escherichia coli was used as a target organism to engineer the 1,6-hexanediol production. The expression strains were obtained after co-transforming all two plasmids in electro competent E. coli MG1655 (DE3) Δrne131, ΔldhA.

Plasmid 1 (ColE1 replicon, ampicillin marker): Gene 1. Plasmid 2 (COLA replicon, kanamycin marker): Gene 2, and Gene 3

(ii) Cell Culturing, Protein Expression, and HDO Production Analysis:

Starter cultures were grown overnight in tubes containing 10 mL LB media with appropriate antibiotics. Cell cultures for the expression and HDO production were carried out in 100 mL volume using glass bottles. Complex growth medium was used and supplemented with 2 g/L D-glucose, 0.5 g/L potassium phosphate buffer (pH 7.2), and other substrates/nutrients important for enzyme expression. Pre-induction growth was carried out for ˜2 hours under aerobic conditions and at 30° C. Recombinant protein expression was induced at an OD600 of 0.2-0.4 with 250 μM IPTG. Post-induction expression was carried out at 30° C. under aerobic conditions for 60-90 minutes followed by 2-3 hours of anaerobic conditions. Afterwards, cells were harvested, concentrated, and re-suspended in 0.5 ml volume at OD600 of ˜40 in fresh medium containing ˜10 g/L glucose, 6-hydroxy-hexanoate (˜5 g/L), and 15 g/L potassium phosphate buffer (pH 7.2). After incubation for 24 hours at room temperature, the cells were centrifuged, and supernatant was filtered and analyzed via HPLC.

(iii) HPLC analysis of HDO production: Isocratic HPLC was used to detect and quantify HDO. The method employed a Bio-Rad Aminex HPX-87 column, 0.7 mL/min of 0.5% formic acid (or 5 mM sulfuric acid) at 35° C. Detection was carried out using an RID (refractive index detector) and UV detector, the latter of which was typically used to measure at signals at 210, 260, and 280 nm. The results showed production of 0.1 to 2.5 g/L of 1,6-hexanediol for all examples in Table 15.

Example 7: Preparation and Use of Microbial Organism for Production of 1,6-Hexanediol from 6-hydroxy-2-keto-hexanoate Intermediate

In some embodiments, the present disclosure provides technologies for preparing 6HH and HDO. In some embodiments, a yield is about or at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 mg/L, or is about or at least about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.7, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 250, or 300 g/L.A biosynthetic pathway for the production of 1,6-hexanediol from 6-hydroxy-2-keto-hexanoate intermediate is shown in FIG. 3. Shown below are examples incorporating the use of different enzymes for each step of this pathway to validate the production of 1,6-hexanediol via this pathway. Examples of genes and corresponding enzymes from which they are encoded that were used to carry out each step of the 1,6-hexanediol biosynthetic pathway from 6-hydroxy-2-keto-hexanoate intermediate are shown in Table 16 below. Each enzyme therein may be substituted with homologous enzymes that belong to the same E.C. class. Additionally, the example below highlights the confirmation of multiple enzymes for carrying out both the CoA-transfer reaction and the 2,6-dihidroxy-hexanoyl-CoA dehydration reaction.

(i) Preparation of Plasmids for HDO Production:

TABLE 16 Biosynthesis polypeptides for HDO production. Example 7A Example 7B Example 7C Example 7D Example 7E Gene Uniprot ID or Uniprot ID or Uniprot ID or Uniprot ID or Uniprot ID or Reaction Catalyzed Enzyme Name Number Genbank ID Genbank ID Genbank ID Genbank ID Genbank ID 6-hydroxy-2-oxohexanoate- 6-hydroxy-2- Gene 1 Q5FTU6 Q5FTU6 Q5FTU6 Q5FTU6 Q5FTU6 → 2,6-dihydroxy- oxohexanoate 2- hexanoate reductase 2,6-dihydroxy-hexanoate → 2,6-dihydroxy- Gene 2 and T4VW93* T4VW93** T4VW93 + T4VW93 + T4VW93 + 2,6-dihydroxy-hexanoyl- hexanoate CoA- Gene 3 A0A0C7GD16 A0A175L1W4 A0A2X3BTQ9 CoA transferase 2,6-dihydroxy-hexanoyl- 2,6-dihydroxy- Gene 4 Q5U924 A0A2X3BK09 A0A2X3BK09 A0A2X3BK09 A0A2X3BK09 CoA → 6-hydroxy-2,3- hexanoyl-CoA 2- dehydro-hexanoyl-CoA dehydratase - Subunit A 2,6-dihydroxy- Gene 5 Q5U925 A0A2X3BU19 A0A2X3BU19 A0A2X3BU19 A0A2X3BU19 hexanoyl-CoA 2- dehydratase - Subunit B 2,6-dihydroxy- Gene 6 Q5U923 A0A1V9IXA9 A0A1V9IXA9 A0A1V9IXA9 A0A1V9IXA9 hexanoyl-CoA 2- dehydratase - Subunit C 6-hydroxy-2,3-dehydro- 2,3-dehydro- Gene 7 Q73Q47 Q73Q47 Q73Q47 Q73Q47 Q73Q47 hexanoyl-CoA → hexanoyl-CoA 6-hydroxy-hexanoyl-CoA 2,3-reductase 6-hydroxy-hexanoyl-CoA → 6-hydroxyhexanoyl- Gene 8 Same as Same as Same as Same as Same as 6-hydroxy-hexanoate CoA transferase Gene 2 & Gene 2 & Gene 2 & Gene 2 & Gene 2 & Gene 3 Gene 3 Gene 4 Gene 5 Gene 6 6-hydroxy-hexanoate → 6-hydroxyhexanoate Gene 9 A0R484 A0R484 A0R484 A0R484 A0R484 6-hydroxy-hexanal 1-reductase 6-hydroxyhexanoate Gene 10 P39135 P39135 P39135 P39135 P39135 1-reductase activator 6-hydroxy-hexanal → 6-hydroxyhexanal Gene 11 AB213459.1 AB213459.1 AB213459.1 AB213459.1 AB213459.1 1,6-hexanediol 1-reductase *single copy of the same gene; **dual copy of the same gene

The HDO production pathway genes were cloned on two separate compatible plasmids shown below. Each plasmid had a different origin of replication and antibiotic marker, as indicated. Synthetic genes were obtained from commercial vendors, and each gene was codon optimized for expression in E. coli. Each gene was cloned under its own T7 promoter and terminator using standard molecular biology methods. Escherichia coli was used as a target organism to engineer the 1,6-hexanediol production. The expression strains were obtained after co-transforming all three plasmids in electro competent E. coli BL21*(DE3) Δldh, ΔadhE, ΔfrdA.

Plasmid 1 (COLA replicon, kanamycin marker): Gene 10, Gene 9,

Plasmid 2 (ColE1 replicon, ampicillin marker): Gene 1, Gene 2, Gene 3, and Gene 4

Plasmid 3 (P15A replicon, chloramphenicol marker): Gene 5, Gene 6, Gene 7, Gene 8, and Gene 11.

(ii) Cell Culturing, Protein Expression, and HDO Production Analysis:

Starter cultures were grown overnight in tubes containing 10 mL LB media with appropriate antibiotics. Cell cultures for the expression and HDO production were carried out in 100 mL volume using glass bottles. Complex growth medium was used and supplemented with 2 g/L D-glucose, 0.5 g/L potassium phosphate buffer (pH 7.2), and other substrates/nutrients important for enzyme expression. Pre-induction growth was carried out for ˜2 hours under aerobic conditions and at 30° C. Recombinant protein expression was induced at an OD600 of 0.2-0.4 with 250 μM IPTG. Post-induction expression was carried out at 30° C. under aerobic conditions for 60-90 minutes followed by 2-3 hours of anaerobic conditions. Afterwards, cells were harvested, concentrated, and re-suspended in 0.5 ml volume at OD600 of ˜40 in fresh medium containing ˜10 g/L glucose, 6-hydroxy-2-keto-hexanoate (˜5 g/L), and 15 g/L potassium phosphate buffer (pH 7.2). After incubation for 24 hours at room temperature, the cells were centrifuged, and supernatant was filtered and analyzed via HPLC.

(iii) HPLC analysis of HDO production: Isocratic HPLC was used to detect and quantify HDO. The method employed a Bio-Rad Aminex HPX-87 column, 0.7 mL/min of 0.5% formic acid (or 5 mM sulfuric acid) at 35° C. Detection was carried out using an RID (refractive index detector) and UV detector, the latter of which was typically used to measure at signals at 210, 260, and 280 nm. The results showed production of 700 mg/L, 1.2 g/L, 1.1 g/L, 1.1 g/L, and 1 g/L of 1,6-hexanediol for Examples 7A-7E from Table 16, respectively.

Example 8: Preparation and Use of Microbial Organism for Production of 1,6-Hexanediol from Different Carbon Sources Via 6-hydroxy-2-keto-hexanoate Intermediate

In some embodiments, the present disclosure provides technologies for preparing 6HH and HDO. In some embodiments, the present disclosure provides technologies for producing HDO using glycerol as a carbon source. In some embodiments, production is carried out in one organism. In some embodiments, production is carried out in two or more organisms each expressing a different set of biosynthesis polypeptides. In some embodiments, production is carried out in a single bacteria strain. In some embodiments, production is carried out in two or more bacteria strains, each independently carrying out one or more biosynthesis reactions. In some embodiments, a culture comprises two or more or all strains for HDO production. In some embodiments, a yield is about or at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 mg/L, or is about or at least about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.7, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 250, or 300 g/L. A biosynthetic pathway for the production of 1,6-hexanediol from pyruvate and 3-hydroxy-propanal through the 6-hydroxy-2-keto-hexanoate intermediate is shown in FIG. 3. Shown below are examples (8a and 8b) incorporating the use of aldolase-hydratase based two enzyme system for production of 1,6-hexanediol via this pathway. A glycerol dehydratase enzyme that is vitamin B12-independent or glycerol dehydratase enzyme that is a B12-dependent enzyme can be cloned to enable production of 3-hydroxy-propionaldehyde—a 1,6-hexanediol pathway precursor that can be made from glycerol using this enzyme. The B12-dependent glycerol dehydratase was used herein. Examples of genes and corresponding enzymes they encode that were used to carry out each step of the 1,6-hexanediol biosynthetic pathway as well as production of 3-hydroxy-propionaldehyde are shown in Table 17. It is important to note that each enzyme herein could be substituted with homologous enzymes that belong to the same E.C class.

TABLE 17 Biosynthesis of HDO. Enzyme Gene Uniprot ID or Reaction Catalyzed Enzyme Name ID Number Genbank ID Pyruvate + 3-hydroxy Trans-o- Ads-Hyd 8 Gene 1 A0A286PH18 propanal → 6-hydroxy-3,4- hydroxybenzylidenepyruvate dehydro-2-oxohexanoate hydratase-aldolases 6-hydroxy-3,4-dehydro-2- Quinone oxidoreductase Qor 1 Gene 2 P28304 oxohexanoate → 6-hydroxy- 2-oxohexanoate 6-hydroxy-2-oxohexanoate- 6-hydroxy-2-oxohexanoate Gene 3 Q5FTU6 → 2,6-dihydroxy-hexanoate 2-reductase 2,6- dihydroxy-hexanoate → 2,6-dihydroxy-hexanoate Gene 4 T4VW93 2,6- dihydroxy-hexanoyl-CoA CoA-transferase 2,6-dihydroxy-hexanoyl-CoA 2,6-dihydroxy-hexanoyl- Gene 5 Q5U924 → 6-hydroxy-2,3-dehydro- CoA 2-dehydratase - hexanoyl-CoA Subunit A 2,6-dihydroxy-hexanoyl- Gene 6 Q5U925 CoA 2-dehydratase - Subunit B 2,6-dihydroxy-hexanoyl- Gene 7 Q5U923 CoA 2-dehydratase - Subunit C 6-hydroxy-2,3-dehydro- 2,3- dehydro-hexanoyl-CoA Gene 8 Q73Q47 hexanoyl-CoA → 6-hydroxy- 2,3- reductase hexanoyl-CoA 6-hydroxy-hexanoyl-CoA → 6-hydroxyhexanoyl-CoA Gene 4 T4VW93 6-hydroxy-hexanoate transferase 6-hydroxy-hexanoate → 6-hydroxyhexanoate 1- Gene 9 A0R484 6-hydroxy-hexanal reductase 6-hydroxyhexanoate 1- Gene 10 P39135 reductase activator 6-hydroxy-hexanal → 6-hydroxyhexanal 1- Gene 11 AB213459.1 1,6-hexanediol reductase Glycerol dehyration Glyerol dehydratase Gene 12 Q8GEZ8 Glyerol dehydratase Gene 13 Q8GEZ7 activator

Example 8a: Production of 1,6-Hexanediol (HDO) in a Single E. coli Strain

(i) Preparation of Plasmids for HDO Production

The HDO production pathway genes were cloned on three separate compatible plasmids shown below. Each plasmid had a different origin of replication and antibiotic marker, as indicated. Synthetic genes were obtained from commercial vendors, and each gene was codon optimized for expression in E. coli. Each gene was cloned under its own T7 promoter and terminator using standard molecular biology methods. Escherichia coli was used as a target organism to engineer the 1,6-hexanediol production. The expression strains were obtained after co-transforming all three plasmids in electro competent E. coli BL21*(DE3) Δldh, ΔadhE, ΔfrdA.

Plasmid 1 (COLA replicon, kanamycin marker): Gene 12, Gene 13, Gene 2, Gene 10

Plasmid 2 (ColE1 replicon, ampicillin marker): Gene 3, Gene 4, Gene 1, and Gene 9

Plasmid 3 (P15A replicon, chloramphenicol marker): Gene 5, Gene 6, Gene 7, Gene 8, and Gene 11.

(ii) Cell Culturing, Protein Expression, and HDO Production Analysis:

Starter cultures were grown overnight in tubes containing 10 mL LB media with appropriate antibiotics. Cell cultures for the expression and HDO production were carried out in 100 mL volume using glass bottles. Complex growth medium was used and supplemented with 2 g/L D-glucose, 0.5 g/L potassium phosphate buffer (pH 7.2), and other substrates/nutrients important for enzyme expression. Pre-induction growth was carried out for ˜2 hours under aerobic conditions and at 30° C. Recombinant protein expression was induced at an OD600 of 0.2-0.4 with 250 μM IPTG. Post-induction expression was carried out at 30° C. under aerobic conditions for 60-90 minutes followed by 2-3 hours of anaerobic conditions. Afterwards, cells were harvested, concentrated, and re-suspended in 0.5 ml volume at OD600 of ˜40 in fresh medium containing 5-20 g/L glucose, 2.5-5 g/L glycerol, and 15 g/L potassium phosphate buffer (pH 7.2). After incubation for 24 hours at room temperature, the cells were centrifuged, and supernatant was filtered and analyzed via HPLC.

(iii) HPLC analysis of HDO production: Isocratic HPLC was used to detect and quantify HDO. The method employed a Bio-Rad Aminex HPX-87 column, 0.7 mL/min of 0.5% formic acid (or 5 mM sulfuric acid) at 35° C. Detection was carried out using an RID (refractive index detector) and UV detector, the latter of which was typically used to measure at signals at 210, 260, and 280 nm. The results showed production of 25-100 mg/L of 1,6-hexanediol. To illustrate that alternate enzymes previously validated to carry out specific steps of the pathway can be used for HDO production using this methodology, an alternate HDO production strain wherein genes 5-7 were encoded by Uniport IDs A0A2X3BK09, A0A2X3BU19, and A0A1V9IXA9 respectively was constructed and evaluated using above methods. This production strain also led to production of >10 mg/L of of 1,6-hexanediol.

Example 8b: Production of 1,6-Hexanediol (HDO) in Two E. coli Strains

(i) Preparation of Plasmids & Strains for HDO Production:

To minimize the number of HDO production pathway genes expressed from plasmids, E. coli expression strain was constructed wherein certain pathway genes were integrated in the genome. Specifically, HDO production strain BL21*(DE3) Δldh, ΔadhE, ΔfrdA containing HDO pathway genes (Gene 12, Gene 13) at the arsB location with expression of each gene controlled by its own T7 promoter. The remaining HDO production pathway genes were cloned on four separate plasmids shown below using techniques described in example above. Identity of Genes was as described in Example 8a. Two E. coli based expression strains were constructed. Expression strain 1 was obtained after co-transforming plasmids 1, and plasmid 2 in E. coli; and Expression strain 2 was obtained after co-transforming plasmid 3 and plasmid 4 in E. coli.

Plasmid 1 (ColE1 replicon, ampicillin marker): Gene 4, gene 3, and gene 1.

Plasmid 2 (P15A replicon, chloramphenicol marker): Gene 5, Gene 6, Gene 7, Gene 8, and Gene 2.

Plasmid 3 (RSF replicon, kanamycin marker): Gene 4, and gene 11.

Plasmid 4 (ColE1 replicon, ampicillin marker): Gene 9 and gene 10.

(ii) Cell Culturing, Protein Expression, and HDO Production Analysis:

Starter cultures were grown overnight in tubes containing 10 mL LB media with appropriate antibiotics for each expression strain separately. Cell cultures for the expression and HDO production were carried out in 100 mL volume using glass bottles for each expression strain separately. Complex growth medium was used and supplemented with 2 g/L D-glucose, 0.5 g/L potassium phosphate buffer (pH 7.2), and other substrates/nutrients important for enzyme expression. Pre-induction growth was carried out for—2 hours under aerobic conditions and at 30° C. for each expression strain separately. Recombinant protein expression was induced at an OD600 of 0.2-0.4 with 250 μM IPTG and was carried out separately for each expression strain. Post-induction expression was carried out at 30° C. under aerobic conditions for 30 minutes followed by 2-3 hours of anaerobic conditions for each expression strain separately. Afterwards, cells from both expression strains were mixed in equal amounts, after which they were harvested, concentrated, and re-suspended in 0.5 ml volume at OD600 of ˜40 in fresh medium containing 5-20 g/L glucose, 2.5-5 g/L glycerol, and 15 g/L potassium phosphate buffer (pH 7.2). After incubation for 24 hours at room temperature, the cells were centrifuged, and supernatant was filtered and analyzed via HPLC.

(iii) HPLC analysis of HDO production: Isocratic HPLC was used to detect and quantify HDO. The method employed a Bio-Rad Aminex HPX-87 column, 0.7 mL/min of 0.5% formic acid (or 5 mM sulfuric acid) at 35° C. Detection was carried out using an RID (refractive index detector) and UV detector, the latter of which was typically used to measure at signals at 210, 260, and 280 nm. The results showed production of 100-550 mg/L of 1,6-hexanediol.

Example 9: Preparation and Use of Microbial Organism for Production of 6-hydroxyhexanoate from 6-hydroxy-2-keto-hexanoate Intermediate

In some embodiments, the present disclosure provides technologies for preparing 6HH. In some embodiments, production is carried out in one organism. In some embodiments, production is carried out in two or more organisms each expressing a different set of biosynthesis polypeptides. In some embodiments, production is carried out in a single bacteria strain. In some embodiments, production is carried out in two or more bacteria strains, each independently carrying out one or more biosynthesis reactions. In some embodiments, a culture comprises two or more or all strains for 6HH production. In some embodiments, a yield is about or at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 mg/L, or is about or at least about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.7, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 250, or 300 g/L 6HH. A biosynthetic pathway for the production of 6-hydroxyhexanoate (6HH) from 6-hydroxy-2-keto-hexanoate intermediate is shown in FIG. 4. Shown below are examples incorporating the use of different enzymes for each step of this pathway to validate the production of 6HH via this pathway. Examples of genes and corresponding enzymes from which they are encoded that were used to carry out each step of the 6HH biosynthetic pathway from 6-hydroxy-2-keto-hexanoate intermediate are shown in Table 18. Each enzyme therein may be substituted with homologous enzymes that belong to the same E.C. class. Additionally, the example below highlights the confirmation of multiple enzymes for carrying out both the CoA-transfer reaction and the 2,6-dihidroxy-hexanoyl-CoA dehydration reaction.

(i) Preparation of Plasmids for 6HH Production:

The 6HH production pathway genes were cloned on two separate compatible plasmids shown below. Each plasmid had a different origin of replication and antibiotic marker, as indicated. Synthetic genes were obtained from commercial vendors, and each gene was codon optimized for expression in E. coli. Each gene was cloned under its own T7 promoter and terminator using standard molecular biology methods. Escherichia coli was used as a target organism to engineer the 6HH production. The expression strains were obtained after co-transforming all three plasmids in electro competent E. coli BL21*(DE3) Δldh, ΔadhE, ΔfrdA.

Plasmid 1 (ColE1 replicon, ampicillin marker): Gene 1, Gene 2, and Gene 3 (only examples 6 & 7)

Plasmid 3 (P15A replicon, chloramphenicol marker): Gene 4, Gene 5, Gene 6, and Gene 7.

TABLE 18 Biosynthesis polypeptides for 6HH production. Gene Example 9A Example 9B Example 9C Example 9D Enzyme Name Number Uniprot ID Uniprot ID Uniprot ID Uniprot ID 6-hydroxy-2-oxohexanoate 2- Gene 1 Q5FTU6 Q5FTU6 Q5FTU6 Q5FTU6 reductase 2,6-dihydroxy-hexanoate CoA- Gene 2 and A0A2X3BTQ9 A0A2X3BTQ9 A0A0C7GD16 T4VW93 transferase Gene 3* 2,6-dihydroxy-hexanoyl-CoA Gene 4 Q5U924 A0A2X3BK09 A0A2X3BK09 A0A2X3BK09 2-dehydratase -Subunit A 2,6-dihydroxy-hexanoyl-CoA Gene 5 Q5U925 A0A2X3BU19 A0A2X3BU19 A0A2X3BU19 2-dehydratase -Subunit B 2,6-dihydroxy-hexanoyl-CoA Gene 6 Q5U923 A0A1V9IXA9 A0A1V9IXA9 A0A1V9IXA9 2-dehydratase -Subunit C 2,3-dehydro-hexanoyl-CoA 2,3- Gene 7 Q73Q47 Q73Q47 Q73Q47 Q73Q47 reductase 6-hydroxyhexanoyl-CoA Gene 2 and A0A2X3BTQ9 A0A2X3BTQ9 A0A0C7GD16 T4VW93 transferase Gene 3* Example 9E Example 9F Example 9G Enzyme Name Uniprot ID Uniprot ID Uniprot ID 6-hydroxy-2-oxohexanoate 2- Q5FTU6 Q5FTU6 A0A1V9IP73 reductase 2,6-dihydroxy-hexanoate CoA- A0A175L1W4 T4VW93 + T4VW93 + transferase A0A175L1W4* A0A175L1W4* 2,6-dihydroxy-hexanoyl-CoA A0A2X3BK09 A0A2X3BK09 A0A2X3BK09 2-dehydratase -Subunit A 2,6-dihydroxy-hexanoyl-CoA A0A2X3BU19 A0A2X3BU19 A0A2X3BU19 2-dehydratase -Subunit B 2,6-dihydroxy-hexanoyl-CoA A0A1V9IXA9 A0A1V9IXA9 A0A1V9IXA9 2-dehydratase -Subunit C 2,3-dehydro-hexanoyl-CoA 2,3- Q73Q47 Q73Q47 Q73Q47 reductase 6-hydroxyhexanoyl-CoA A0A175L1W4 T4VW93 + T4VW93 + transferase A0A175L1W4 A0A175L1W4 *present only for Examples 9F and 9G

(ii) Cell Culturing, Protein Expression, and 6HH Production Analysis:

Starter cultures were grown overnight in tubes containing 10 mL LB media with appropriate antibiotics. Cell cultures for the expression and 6HH production were carried out in 100 mL volume using glass bottles. Complex growth medium was used and supplemented with 2 g/L D-glucose, 0.5 g/L potassium phosphate buffer (pH 7.2), and other substrates/nutrients important for enzyme expression. Pre-induction growth was carried out for ˜2 hours under aerobic conditions and at 30° C. Recombinant protein expression was induced at an OD600 of 0.2-0.4 with 250 μM IPTG. Post-induction expression was carried out at 30° C. under aerobic conditions for 60-90 minutes followed by 2-3 hours of anaerobic conditions. Afterwards, cells were harvested, concentrated, and re-suspended in 0.5 ml volume at OD600 of ˜40 in fresh medium containing ˜10 g/L glucose, 6-hydroxy-2-keto-hexanoate (5-10 g/L), and 15 g/L potassium phosphate buffer (pH 7.2). After incubation for 24 hours at room temperature, the cells were centrifuged, and supernatant was filtered and analyzed via HPLC.

(iii) HPLC analysis of HDO production: Isocratic HPLC was used to detect and quantify HDO. The method employed a Bio-Rad Aminex HPX-87 column, 0.7 mL/min of 0.5% formic acid (or 5 mM sulfuric acid) at 35° C. Detection was carried out using an RID (refractive index detector) and UV detector, the latter of which was typically used to measure at signals at 210, 260, and 280 nm. The results showed production of ˜0.4-5 g/L of 6HH from strains of Examples 9A-9G of Table 18.

Example 10: Preparation and Use of Microbial Organism for Production of 6-hydroxy hexanoic acid (6HH) from Different Carbon Sources via 6-hydroxy-2-keto-hexanoate Intermediate

In some embodiments, the present disclosure provides technologies for preparing 6HH. In some embodiments, the present disclosure provides technologies for producing 6HH using glycerol as a carbon source. In some embodiments, production is carried out in one organism. In some embodiments, production is carried out in two or more organisms each expressing a different set of biosynthesis polypeptides. In some embodiments, production is carried out in a single bacteria strain. In some embodiments, production is carried out in two or more bacteria strains, each independently carrying out one or more biosynthesis reactions. In some embodiments, a culture comprises two or more or all strains for 6HH production. In some embodiments, a yield is about or at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 mg/L, or is about or at least about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.7, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 250, or 300 g/L. A biosynthetic pathway for the production of 6HH from pyruvate and 3-hydroxy-propanal through the 6-hydroxy-2-keto-hexanoate intermediate is shown in FIG. 4. Shown below are examples incorporating the use of aldolase-hydratase based two enzyme system for production of 6HH via this pathway. A glycerol dehydratase enzyme that is vitamin B12-independent or glycerol dehydratase enzyme that is a B12-dependent enzyme can be cloned to enable production of 3-hydroxy-propionaldehyde—a 6HH pathway precursor that can be made from glycerol using this enzyme. Although both types of glycerol dehydratases were used herein, entries shown in Table 19 focus on examples that use the B12-independent glycerol dehydratase enzyme. Each enzyme therein may be substituted with homologous enzymes that belong to the same E.C. class to yield 6HH, and Examples 10B and 10C in Table 19 demonstrate this point wherein enzymes catalyzing both CoA-transfer reactions and the 2,6-dihidroxy-hexanoyl-CoA dehydration reactions have been substituted with homologous enzymes.

(i) Preparation of Plasmids & Strains for 6HH Production:

To minimize the number of 6HH production pathway genes expressed from plasmids, E. coli expression strain was constructed wherein certain pathway genes were integrated in the genome. Specifically, 6HH production strain BL21*(DE3) Δldh, ΔadhE, ΔfrdA containing 6HH pathway genes (Gene 12, Gene 13) at the arsB location with expression of each gene controlled by its own T7 promoter. The remaining 6HH production pathway genes were cloned on two separate plasmids shown below using techniques described in example above.

Plasmid 1 (ColE1 replicon, ampicillin marker): Gene 4, gene 3, and gene 1.

Plasmid 2 (P15A replicon, chloramphenicol marker): Gene 5, Gene 6, Gene 7, Gene 8, and Gene 2.

(ii) Cell Culturing, Protein Expression, and 6HH Production Analysis:

Starter cultures were grown overnight in tubes containing 10 mL LB media with appropriate antibiotics for each expression strain separately. Cell cultures for the expression and HDO production were carried out in 100 mL volume using glass bottles for each expression strain separately. Complex growth medium was used and supplemented with 2 g/L D-glucose, 0.5 g/L potassium phosphate buffer (pH 7.2), and other substrates/nutrients important for enzyme expression. Pre-induction growth was carried out for ˜2 hours under aerobic conditions and at 30° C. for each expression strain separately. Recombinant protein expression was induced at an OD600 of 0.2-0.4 with 250 μM IPTG and was carried out separately for each expression strain. Post-induction expression was carried out at 30° C. under aerobic conditions for 30 minutes followed by 2-3 hours of anaerobic conditions for each expression strain separately. Afterwards, cells were harvested, concentrated, and re-suspended in 0.5 ml volume at OD600 of ˜40 in fresh medium containing 5-20 g/L glucose, 2.5-5 g/L glycerol, and 15 g/L potassium phosphate buffer (pH 7.2). After incubation for 24 hours at room temperature, the cells were centrifuged, and supernatant was filtered and analyzed via HPLC.

TABLE 19 Biosynthesis polypeptides for 6HH. Example 10A Example 10B Example 10C Gene Uniprot ID or Uniprot ID or Uniprot ID or Reaction Catalyzed Enzyme Name Enzyme ID Number Genbank ID Genbank ID Genbank ID Pyruvate + 3-hydroxy Trans-o- Ads-Hyd 8 Gene 1 A0A286PH18 A0A286PH18 A0A286PH18 propanal → 6-hydroxy-3,4- hydroxybenzylidenepyruvate dehydro-2-oxohexanoate hydratase -aldolases 6-hydroxy-3,4-dehydro-2- Quinone oxidoreductase Qor-1 Gene 2 P28304 P28304 P28304 oxohexanoate → 6-hydroxy- 2-oxohexanoate 6-hydroxy-2-oxohexanoate 6-hydroxy-2-oxohexanoate 2- Gene 3 Q5FTU6 Q5FTU6 Q5FTU6 → 2,6-dihydroxy-hexanoate reductase 2,6-dihydroxy-hexanoate → 2,6-dihydroxy-hexanoate Gene 4 T4VW93 A0A2X3BTQ9 T4VW93 2,6-dihydroxy-hexanoyl-CoA CoA-transferase 2,6-dihydroxy-hexanoyl-CoA 2,6-dihydroxy-hexanoyl-CoA Gene 5 Q5U924 A0A2X3BK09 A0A2X3BK09 → 6-hydroxy-2,3-dehydro- 2-dehydratase -Subunit A hexanoyl-CoA 2,6-dihydroxy-hexanoyl-CoA Gene 6 Q5U925 A0A2X3BU19 A0A2X3BU19 2-dehydratase -Subunit B 2,6-dihydroxy-hexanoyl-CoA Gene 7 Q5U923 A0A1V9IXA9 A0A1V9IXA9 2-dehydratase -Subunit C 6-hydroxy-2,3-dehydro- 2,3-dehydro-hexanoyl-CoA Gene 8 Q73Q47 Q73Q47 Q73Q47 hexanoyl-CoA → 6-hydroxy- 2,3-reductase hexanoyl-CoA 6-hydroxy-hexanoyl-CoA → 6-hydroxyhexanoyl-CoA Gene 4 T4VW93 A0A2X3BTQ9 T4VW93 6-hydroxy-hexanoate transferase Glycerol dehydration Glycerol dehydratase Gene 12 Q8GEZ8 Q8GEZ8 Q8GEZ8 Glycerol dehydratase Gene 13 Q8GEZ7 Q8GEZ7 Q8GEZ7 activator

(iii) HPLC analysis of 6HH production: Isocratic HPLC was used to detect and quantify HDO. The method employed a Bio-Rad Aminex HPX-87 column, 0.7 mL/min of 0.5% formic acid (or 5 mM sulfuric acid) at 35° C. Detection was carried out using an RID (refractive index detector) and UV detector, the latter of which was typically used to measure at signals at 210, 260, and 280 nm. The results showed production of ˜50-800 mg/L of 6HH from strains of Examples 10A-10C in Table 19. An alternative example is where B12-dependent glycerol dehydratase pduCDEGH was used (encoded as a single gene operon on a third plasmid with COLA replicon, kanamycin marker) instead of B12-independent glycerol dehydratase, wherein the rest of the enzymes of the pathway were identical to Example 10A. Such a system also led to production of ˜350 mg/L of 6HH using culture conditions described for strains PeDO3 and PeDO4 containing B12-dependent enzymes in Example 5.

Example 11: Preparation and Use of Microbial Organism for Production of Adipic acid (AA) from 6-hydroxy-hexanoate (6HH) Intermediate

In some embodiments, the present disclosure provides technologies for preparing AA. In some embodiments, a yield is about or at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 mg/L, or is about or at least about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.7, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 250, or 300 g/L. A biosynthetic pathway for the production of AA from pyruvate and 3-hydroxy-propanal through the 6-hydroxy-hexanoate intermediate is shown in FIG. 5. Shown in Table 20 are examples of enzymes that enable the conversion of 6HH to AA. It is important to note that each enzyme herein could be substituted with homologous enzymes that belong to the same E.C class to yield AA.

TABLE 20 Biosynthesis polypeptides for AA. Example 11A Example 11B Reaction Gene Uniprot ID or Uniprot ID or Catalyzed Enzyme Name Number Genbank ID Genbank ID 6-hydroxy- 6-hydroxy- Gene 1 Q7WVD0 Q84H78 hexanoate → hexanoate 6-oxo- dehydrogenase hexanoate 6-oxo- 6-oxo- Gene 2 Q9R2F4 Q9R2F4 hexanoate → hexanoate Adipic acid oxidase

(i) Preparation of plasmids & strains for AA production from 6HH: The AA production pathway genes were cloned on a single plasmid shown below using techniques described in examples before. BL21*(DE3) Δldh, ΔadhE, ΔfrdA was used as the production strain.

Plasmid 1 (ColE1 replicon, ampicillin marker): Gene 1, and gene 2.

(ii) Cell culturing, protein expression, and AA production analysis: Starter cultures were grown overnight in tubes containing 10 mL LB media with appropriate antibiotics for each expression strain separately. Cell cultures for the expression and AA production were carried out in 100 mL volume using glass bottles for each expression strain separately. Complex growth medium was used and supplemented with 2 g/L D-glucose, 0.5 g/L potassium phosphate buffer (pH 7.2), and other substrates/nutrients important for enzyme expression. Pre-induction growth was carried out for ˜2 hours under aerobic conditions and at 30° C. for each expression strain separately. Recombinant protein expression was induced at an OD600 of 0.2-0.4 with 250 μM IPTG and was carried out separately for each expression strain. Post-induction expression was carried out at 30° C. under aerobic conditions for 30-120 minutes followed by 2-3 hours of anaerobic conditions for each expression strain separately. Afterwards, cells were harvested, concentrated, and re-suspended in 0.5 ml volume at OD600 of ˜40 in fresh medium containing 5-10 g/L glucose, 5 g/L 6HH, and 15 g/L potassium phosphate buffer (pH 7.2). After incubation for 3 hours at room temperature, the cells were centrifuged, and supernatant was filtered and analyzed via HPLC.

(iii) HPLC analysis of AA production: Isocratic HPLC was used to detect and quantify AA. The method employed a Bio-Rad Aminex HPX-87 column, 0.7 mL/min of 0.5% formic acid (or 5 mM sulfuric acid) at 35° C. Detection was carried out using an RID (refractive index detector) and UV detector, the latter of which was typically used to measure at signals at 210, 260, and 280 nm. The results showed production of 500-1500 mg/L of AA for Examples 11A and 11B of Table 20.

Example 12: Preparation and Use of Microbial Organism for Production of Adipic acid (AA) from 6-hydroxy-2-keto-hexanoate Intermediate

In some embodiments, the present disclosure provides technologies for preparing AA from 6H2KH. In some embodiments, a yield is about or at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 mg/L, or is about or at least about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.7, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 250, or 300 g/L. A biosynthetic pathway for the production of AA from pyruvate and 3-hydroxy-propanal through the 6-hydroxy-2-keto-hexanoate intermediate is shown in FIG. 5. Shown below are examples incorporating the use of different enzymes for each step of this pathway to validate the production of AA via this pathway. Examples of genes and corresponding enzymes from which they are encoded that were used to carry out each step of the AA biosynthetic pathway from 6-hydroxy-2-keto-hexanoate intermediate are shown in Table 21 below. Each enzyme therein may be substituted with homologous enzymes that belong to the same E.C. class. Examples 12A and 12B in Table 21 highlight the confirmation of multiple enzymes for carrying out both CoA-transfer reaction and the 2,6-dihidroxy-hexanoyl-CoA dehydration reaction to enable successful production of AA via this pathway.

(i) Preparation of plasmids & strains for AA production from 6-hydroxy-2-keto-hexanoate: The AA production pathway genes were cloned on two separate compatible plasmids shown below. Each plasmid had a different origin of replication and antibiotic marker, as indicated. Synthetic genes were obtained from commercial vendors, and each gene was codon optimized for expression in E. coli. Each gene was cloned under its own T7 promoter and terminator using standard molecular biology methods. Escherichia coli was used as a target organism to engineer the 6HH production. The expression strains were obtained after co-transforming both plasmids in electro competent E. coli MG1655 (DE3) me 131 ΔldhA ΔadhE ΔfrdBC.

Plasmid 1 (ColE1 replicon, ampicillin marker): Gene 3, Gene 4, Gene 9, and Gene 10

Plasmid 3 (P15A replicon, chloramphenicol marker): Gene 5, Gene 6, Gene 7, and Gene 8

(ii) Cell culturing, protein expression, and AA production analysis: Same as example 11 except 10 g/L 6-hydroxy-2-keto-hexanoate was used (instead of 6HH used in example 11) as the substrate.

(iii) HPLC analysis of AA production: Isocratic HPLC was used to detect and quantify AA as described above. The results showed production of 100-800 mg/L of AA for Examples 12A-12C of Table 21.

TABLE 21 Biosynthesis polypeptides for AA. Example 12A Example 12B Example 12C Gene Uniprot ID or Uniprot ID or Uniprot ID or Reaction Catalyzed Enzyme Name Number Genbank ID Genbank ID Genbank ID 6-hydroxy-2-oxohexanoate → 2,6- 6-hydroxy-2-oxohexanoate 2- Gene 3 Q5FTU6 Q5FTU6 Q5FTU6 dihydroxy-hexanoate reductase 2,6-dihydroxy-hexanoate → 2,6- 2,6-dihydroxy-hexanoate CoA- Gene 4 T4VW93 A0A2X3BTQ9 T4VW93 dihydroxy-hexanoyl-CoA transferase 2,6-dihydroxy-hexanoyl-CoA → 2,6-dihydroxy-hexanoyl-CoA 2- Gene 5 Q5U924 A0A2X3BK09 A0A2X3BK09 6-hydroxy-2,3-dehydro-hexanoyl- dehydratase -Subunit A CoA 2,6-dihydroxy-hexanoyl-CoA 2- Gene 6 Q5U925 A0A2X3BU19 A0A2X3BU19 dehydratase -Subunit B 2,6-dihydroxy-hexanoyl-CoA 2- Gene 7 Q5U923 A0A1V9IXA9 A0A1V9IXA9 dehydratase -Subunit C 6-hydroxy-2,3-dehydro-hexanoyl- 2,3-dehydro-hexanoyl-CoA 2,3- Gene 8 Q73Q47 Q73Q47 Q73Q47 CoA → 6-hydroxy-hexanoyl-CoA reductase 6-hydroxy-hexanoyl-CoA → 6- 6-hydroxyhexanoyl-CoA Gene 4 T4VW93 A0A2X3BTQ9 T4VW93 hydroxy-hexanoate transferase 6-hydroxy-hexanoate → 6-oxo- 6-hydroxyhexanoate Gene 9 Q84H78 Q84H78 Q84H78 hexanoate dehydrogenase 6-oxo-hexanoate → Adipic acid 6-oxo-hexanoate oxidase Gene 10 Q9R2F4 Q9R2F4 Q9R2F4

Example 13: Preparation and Use of Microbial Organism for Production of Adipic acid (AA) from Different Carbon Sources via 6-hydroxy-2-keto-hexanoate Intermediate

In some embodiments, the present disclosure provides technologies for preparing AA. In some embodiments, the present disclosure provides technologies for producing AA using 3HPA and pyruvate. In some embodiments, the present disclosure provides technologies for producing AA using glycerol as a carbon source. In some embodiments, production is carried out in one organism. In some embodiments, production is carried out in two or more organisms each expressing a different set of biosynthesis polypeptides. In some embodiments, production is carried out in a single bacteria strain. In some embodiments, production is carried out in two or more bacteria strains, each independently carrying out one or more biosynthesis reactions. In some embodiments, a culture comprises two or more or all strains for AA production. In some embodiments, a yield is about or at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 mg/L, or is about or at least about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.7, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 250, or 300 g/L. A biosynthetic pathway for the production of AA from pyruvate and 3-hydroxy-propanal through the 6-hydroxy-2-keto-hexanoate intermediate is shown in FIG. 5. Shown below are examples incorporating the use of aldolase-hydratase-based two-enzyme system for production of AA via this pathway. A glycerol dehydratase enzyme that is vitamin B12-independent or a glycerol dehydratase enzyme that is a B12-dependent enzyme can be cloned to enable production of 3-hydroxy-propionaldehyde—a 6HH pathway precursor that can be made from glycerol using this enzyme. The B12-dependent glycerol dehydratase was used herein. Examples of genes and corresponding enzymes they encode that were used to carry out each step of AA biosynthetic pathway as well as production of 3-hydroxy-propionaldehyde are shown in Table 22. Each enzyme therein may be substituted with homologous enzymes that belong to the same E.C. class.

TABLE 22 Biosynthesis polypeptides for AA. Gene Uniprot ID or Reaction Catalyzed Enzyme Name Enzyme ID Number Genbank ID Pyruvate + 3-hydroxy Trans-o- Ads-Hyd 8 Gene 1 A0A286PH18 propanal → 6-hydroxy-3,4- hydroxybenzylidenepyruvate dehydro-2-oxohexanoate hydratase-aldolases 6-hydroxy-3,4-dehydro-2- Quinone oxidoreductase Qor 1 Gene 2 P28304 oxohexanoate → 6- hydroxy-2-oxohexanoate 6-hydroxy-2-oxohexanoate 6-hydroxy-2-oxohexanoate Gene 3 Q5FTU6 → 2,6-dihydroxy- 2-reductase hexanoate 2,6-dihydroxy-hexanoate 2,6-dihydroxy-hexanoate Gene 4 T4VW93 → 2,6-dihydroxy- CoA-transferase hexanoyl-CoA 2,6-dihydroxy-hexanoyl- 2,6-dihydroxy-hexanoyl- Gene 5 Q5U924 CoA → 6-hydroxy-2,3- CoA 2-dehydratase -Subunit A dehydro-hexanoyl-CoA 2,6-dihydroxy-hexanoyl- Gene 6 Q5U925 CoA 2-dehydratase -Subunit B 2,6-dihydroxy-hexanoyl- Gene 7 Q5U923 CoA 2-dehydratase -Subunit C 6-hydroxy-2,3-dehydro- 2,3-dehydro-hexanoyl-CoA Gene 8 Q73Q47 hexanoyl-CoA → 2,3-reductase 6-hydroxy-hexanoyl-CoA 6-hydroxy-hexanoyl-CoA 6-hydroxyhexanoyl-CoA Gene 4 T4VW93 → 6-hydroxy-hexanoate transferase 6-hydroxy-hexanoate → 6-hydroxyhexanoate Gene 9 Q84H78 6-oxo-hexanoate dehydrogenase 6-oxo-hexanoate → 6-oxo-hexanoate oxidase Gene 10 Q9R2F4 Adipic acid Glycerol dehydration Glycerol dehydratase Gene 12 Q8GEZ8 Glycerol dehydratase Gene 13 Q8GEZ7 activator

(i) Preparation of Plasmids & Strains for AA Production:

To minimize the number of AA production pathway genes expressed from plasmids, E. coli expression strain was constructed wherein certain pathway genes were integrated in the genome. Specifically, AA production strain BL21*(DE3) Δldh, ΔadhE, ΔfrdA containing pathway genes (Gene 12, Gene 13) at the arsB location with expression of each gene controlled by its own T7 promoter. Two E. coli based expression strains were constructed. Expression strain 1 was obtained after co-transforming plasmids 1, and plasmid 2 in E. coli; and Expression strain 2 was obtained after transforming plasmid 3 in E. coli.

Plasmid 1 (ColE1 replicon, ampicillin marker): Gene 4, gene 3, and gene 1.

Plasmid 2 (P15A replicon, chloramphenicol marker): Gene 5, Gene 6, Gene 7, Gene 8, and Gene 2.

Plasmid 3 (ColE1 replicon, ampicillin marker): Gene 9, gene 10, and gene 3.

(ii) Cell Culturing, Protein Expression, and AA Production Analysis:

Starter cultures were grown overnight in tubes containing 10 mL LB media with appropriate antibiotics for each expression strain separately. Cell cultures for the expression and AA production were carried out in 100 mL volume using glass bottles for each expression strain separately. Complex growth medium was used and supplemented with 2 g/L D-glucose, 0.5 g/L potassium phosphate buffer (pH 7.2), and other substrates/nutrients important for enzyme expression. Pre-induction growth was carried out for ˜2 hours under aerobic conditions and at 30° C. for each expression strain separately. Recombinant protein expression was induced at an OD600 of 0.2-0.4 with 250 μM IPTG and was carried out separately for each expression strain. Post-induction expression was carried out at 30° C. under aerobic conditions for 30 minutes followed by 2-3 hours of anaerobic conditions for each expression strain separately. Afterwards, cells were harvested, concentrated, and re-suspended in 0.5 ml volume at OD600 of ˜40 in fresh medium containing 5-20 g/L glucose, 2.5-5 g/L glycerol, and 15 g/L potassium phosphate buffer (pH 7.2). After incubation for 24 hours at room temperature, the cells were centrifuged, and supernatant was filtered and analyzed via HPLC.

(iii) HPLC analysis of AA production: Isocratic HPLC was used to detect and quantify AA. The method employed a Bio-Rad Aminex HPX-87 column, 0.7 mL/min of 0.5% formic acid (or 5 mM sulfuric acid) at 35° C. Detection was carried out using an RID (refractive index detector) and UV detector, the latter of which was typically used to measure at signals at 210, 260, and 280 nm. The results showed production of 20-350 mg/L of AA.

Example 14: Multi-strain and multi-pot production of 6-hydroxyhexanoate

In some embodiments, production of a product e.g., 6HH, is carried out in one strain. In some embodiments, production is carried out in two or more strains. In some embodiments, the two or more strains together express all biosynthesis polypeptides utilized in a production. In some embodiments, a product of a biosynthesis polypeptide in one strain is a substrate of a biosynthesis polypeptide of another strain. In some embodiments, products of two or more biosynthesis polypeptides of one strain are independently substrates of two or more biosynthesis polypeptides in one or more other strains. In some embodiments, a yield is about or at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 mg/L, or is about or at least about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.7, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 250, or 300 g/L of 6-hydroxyhexanoate.

Example 10 above describes the production of a 6HH in a single E. coli strain, wherein all the biosynthetic pathway enzymes necessary for converstion of pyruvate and 3-hydroxy propanal (and its production from glycerol) are all expressed simultaneously within a single E. coli strain. In some embodiments, it might be advantageous to pursue a multistrain approach, wherein the entire biosynthetic pathway is split into smaller sections called modules, wherein each module comprises a series of sequential enzymes of the biosynthetic pathway that are expressed in its own unique E. coli strain. For example, it was demonstrated that it was feasible to split the entire 6HH biosynthetic pathway into two modules. Specifically, described in Example 3 above is a construction of the first module, which allows for production of 6-hydroxy-2-keto-hexanoate—an intermediate of the 6HH biosynthetic pathway in a single E. coli strain, wherein all enzymes necessary for conversion of pyruvate and 3-hydroxy propanal (and its production from glycerol) were all expressed simultaneously within a single E. coli strain. Described in Example 9 above is a construction of the second module, which allows for production of 6HH from 6-hydroxy-2-keto-hexanoate in a second (separate) E. coli strain, wherein all enzymes necessary for conversion of 6-hydroxy-2-keto-hexanoate to 6HH are all expressed simultaneously within this single E. coli strain. Use of both modules leads to a complete biosynthetic pathway for production of 6HH in two separate E. coli strains. Such a multistrain approach can be advantageous for a number of reasons such as, but not limited to: a) constructing and testing plasmids for developing extensive biosynthetic pathways like these can result in large libraries, and conventional brute-force methods of screening for functional (or the best) genetic constructs can be inefficient and expensive; b) enzyme expression may be simplified and balanced across the pathway leading to substantially faster development cycles; c) genetic background of E. coli strains for each separate module may be tailored to suit redox, ATP, and other needs to maximize production for each module (since a single strain optimization may not be efficient for the entire pathway). Results summarized in Table 23 below demonstrate the successful use of this multi-strain approach for the production of 6HH either in simultaneous (i.e., one-pot) or via sequential production methodology.

TABLE 23 Production of 6-hydroxyhexanoate. Example 14A Example 14B Growth: multi-pot multi-pot Production: one-pot sequential Titer: 350 mg/L 6HH 1.1 g/L 6HH Gene Uniprot Reaction Catalyzed Number ID Host Pyruvate + 3-hydroxy propanal → 6-hydroxy-3,4- Gene 1 A0A286PH18 strain 1 dehydro-2-oxohexanoate 6-hydroxy-3,4-dehydro-2-oxohexanoate → 6-hydroxy- Gene 2 P28304 strain 1 2-oxohexanoate 6-hydroxy-2-oxohexanoate → 2,6-dihydroxy- Gene 3 Q5FTU6 strain 2 hexanoate 2,6-dihydroxy-hexanoate → 2,6-dihydroxy-hexanoyl- Gene 4 A0A2X3BTQ9 strain 2 CoA 2,6-dihydroxy-hexanoyl-CoA → 6-hydroxy-2,3- Gene 5 A0A2X3BK09 strain 2 dehydro-hexanoyl-CoA Gene 6 A0A2X3BU19 strain 2 Gene 7 A0A1V9IXA9 strain 2 6-hydroxy-2,3-dehydro-hexanoyl-CoA → 6-hydroxy- Gene 8 Q73Q47 strain 2 hexanoyl-CoA 6-hydroxy-hexanoyl-CoA → 6-hydroxy-hexanoate Gene 4 A0A2X3BTQ9 strain 2 Glyerol dehydratase (B12-dependent) Gene 9 Lre PduCDEGH* strain 1 *Lre PduCDEGH is a vitamin B-12 dependent glycerol dehydratase and its corresponding activator from Lactococcus reuteri. It is encoded by a single gene operon encoded that is comprised of five genes as follows: pduC [Uniprot ID No. A5VMB2]; pduD [Uniprot ID No. A5VMB1]; pduE [Uniprot ID No. A5VMB0]; pduG [Uniprot ID No. A5VMA9]; and pduH [Uniprot ID No. A5VMA8]).

(i) Preparation of Plasmids & Strains for 6HH Production:

The entire 6HH biosynthetic pathway was split into two E. coli strains (or modules) as described above. Two E. coli based expression strains were constructed. Expression strain 1 was obtained after co-transforming plasmids 1, and plasmid 2 in E. coli MG1655 (DE3) rne131 ΔldhA ΔadhE ΔfrdBC ΔpoxB ΔpflB ΔackA-pta ΔyqhD, ΔadhP, ΔeutG, ΔgldA, ΔyiaY, ΔfucO; and Expression strain 2 was obtained after transforming plasmid 3 and 4 in E. coli MG1655 (DE3) me 131 ΔldhA ΔadhE ΔfrdBC.

Plasmid 1 (ColE1 replicon, ampicillin marker): Gene 1, gene 2, and gene 1.

Plasmid 2 (P15A replicon, chloramphenicol marker): Gene 9.

Plasmid 3 (ColE1 replicon, ampicillin marker): Gene 4.

Plasmid 4 (P15A replicon, chloramphenicol marker): Gene 5, Gene 6, Gene 7, Gene 8, and Gene 3.

(ii) Cell Culturing, Protein Expression, and 6HH Production Analysis:

Starter cultures were grown overnight in tubes containing 10 mL LB media with appropriate antibiotics for each expression strain separately. Cell cultures for the expression and 6HH production were carried out in 100 mL volume using glass bottles for each expression strain separately. Complex growth medium was used and supplemented with 2 g/L D-glucose, 0.5 g/L potassium phosphate buffer (pH 7.2), and other substrates/nutrients important for enzyme expression. Pre-induction growth was carried out for ˜2 hours under aerobic conditions and at 30° C. for each expression strain separately. Recombinant protein expression was induced at an OD600 of 0.2-0.4 with 250 μM IPTG and was carried out separately for each expression strain. Post-induction expression was carried out at 30° C. under aerobic conditions for 30 minutes followed by 2-3 hours of anaerobic conditions for each expression strain separately. Afterwards, cells from both expression strains were harvested, concentrated, and re-suspended in 0.5 ml volume at OD600 of ˜40. For Example 14A, equal number cells from both strains were re-suspended in media containing 5-20 g/L glucose, 2.5-5 g/L glycerol, and 15 g/L potassium phosphate buffer (pH 7.2). After incubation for 24 hours at room temperature, the cells were centrifuged, and supernatant was filtered and analyzed via HPLC. For Example 14B, cells from expression strain 1 was suspended in media containing 5-20 g/L glucose, 2.5-5 g/L glycerol, and 15 g/L potassium phosphate buffer (pH 7.2). After incubation for 24 hours at room temperature, the cells were centrifuged, and supernatant was filtered and mixed with cells from expression strain 2. After incubation for 24 hours at room temperature, the cells were centrifuged, and supernatant was filtered and analyzed by HPLC.

(iii) HPLC analysis of 6HH production: This was carried out as mentioned before. The results showed production of 350-1100 mg/L of 6HH.

Example 15: Multi-Strain and Multi-Pot Production of 1,6-hexanediol

In some embodiments, the present disclosure provides technologies for preparing HDO. In some embodiments, the present disclosure provides technologies for producing HDO from 3HPA and pyruvate. In some embodiments, the present disclosure provides technologies for producing HDO using glycerol as a carbon source. In some embodiments, production is carried out in one organism. In some embodiments, production is carried out in two or more organisms each expressing a different set of biosynthesis polypeptides. In some embodiments, production is carried out in a single bacteria strain. In some embodiments, production is carried out in two or more bacteria strains, each independently carrying out one or more biosynthesis reactions. In some embodiments, a culture comprises two or more or all strains for HDO production. In some embodiments, a yield is about or at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 mg/L, or is about or at least about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.7, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 250, or 300 g/L. Examples 8 above describe the production of HDO in a single or dual E. coli strain, wherein all the biosynthetic pathway enzymes necessary for conversion of pyruvate and 3-hydroxy propanal (and its production from glycerol) are all expressed simultaneously within a single E. coli strain or two separate E. coli strains. Such a multi-strain approach can be advantageous for a number of reasons mentioned in Example 14. Results summarized in Table 24 demonstrate another successful use of this multi-strain approach for the production of HDO either in simultaneous (i.e., one-pot) or via sequential production methodology.

TABLE 24 Production of 1,6-hexanediol. Example 15A Example 15B Growth: multi-pot multi-pot Production: one-pot sequential Titer: 400 mg/L 16HDO 800 mg/L 16HDO Gene Uniprot ID or Reaction Catalyzed Number Genbank ID Host Pyruvate + 3-hydroxy propanal → 6- Gene 1 A0A286PH18 strain hydroxy-3,4-dehydro-2-oxohexanoate 1 6-hydroxy-3,4-dehydro-2-oxohexanoate Gene 2 P28304 strain → 6-hydroxy-2-oxohexanoate 1 6-hydroxy-2-oxohexanoate → 2,6- Gene 3 Q5FTU6 strain dihydroxy-hexanoate 2 2,6-dihydroxy-hexanoate → 2,6- Gene 4 A0A2X3BTQ9 strain dihydroxy-hexanoyl-CoA 2 2,6-dihydroxy-hexanoyl-CoA → 6- Gene 5 A0A2X3BK09 strain hydroxy-2,3-dehydro-hexanoyl-CoA 2 Gene 6 A0A2X3BU19 strain 2 Gene 7 A0A1V9IXA9 strain 2 6-hydroxy-2,3-dehydro-hexanoyl-CoA → Gene 8 Q73Q47 strain 6-hydroxy-hexanoyl-CoA 2 6-hydroxy-hexanoyl-CoA → 6-hydroxy- Gene 4 A0A2X3BTQ9 strain hexanoate 2 6-hydroxy-hexanoate → 6-hydroxy- Gene 9 A0R484 strain hexanal 2 Gene 10 P39135 strain 2 6-hydroxy-hexanal → 1,6-hexanediol Gene 11 AB213459.1 strain 2 Glyerol dehydratase (B12-dependent) Gene 12 Lre PduCDEGH* strain 1 *Lre PduCDEGH is a vitamin B-12 dependent glycerol dehydratase and its corresponding activator from Lactococcus reuteri. It is encoded by a single gene operon encoded that is comprised of five genes as follows: pduC [Uniprot ID No. A5VMB2]; pduD [Uniprot ID No. A5VMB1]; pduE [Uniprot ID No. A5VMB0]; pduG [Uniprot ID No. A5VMA9]; and pduH [Uniprot ID No. A5VMA8]).

(i) Preparation of Plasmids & Strains for HDO Production:

The entire HDO biosynthetic pathway was split into two E. coli strains (or modules) as described above. Two E. coli based expression strains were constructed. Expression strain 1 was obtained after co-transforming plasmids 1, and plasmid 2 in E. coli MG1655 (DE3) rne131 ΔldhA ΔadhE ΔfrdBC ΔpoxB ΔpflB ΔackA-pta ΔyqhD, ΔadhP, ΔeutG, ΔgldA, ΔyiaY, ΔfucO; and Expression strain 2 was obtained after transforming plasmid 3 and 4 in E. coli MG1655 (DE3) me 131 ΔldhA ΔadhE ΔfrdBC.

Plasmid 1 (ColE1 replicon, ampicillin marker): Gene 1, gene 2, and gene 1.

Plasmid 2 (P15A replicon, chloramphenicol marker): Gene 12.

Plasmid 3 (ColE1 replicon, ampicillin marker): Gene 3, Gene 9, Gene 4, Gene 11, and Gene 10.

Plasmid 4 (P15A replicon, chloramphenicol marker): Gene 5, Gene 6, Gene 7, Gene 8, and Gene 4.

(ii) Cell Culturing, Protein Expression, and HDO Production Analysis:

Starter cultures were grown overnight in tubes containing 10 mL LB media with appropriate antibiotics for each expression strain separately. Cell cultures for the expression and 6-HH production were carried out in 100 mL volume using glass bottles for each expression strain separately. Complex growth medium was used and supplemented with 2 g/L D-glucose, 0.5 g/L potassium phosphate buffer (pH 7.2), and other substrates/nutrients important for enzyme expression. Pre-induction growth was carried out for ˜2 hours under aerobic conditions and at 30° C. for each expression strain separately. Recombinant protein expression was induced at an OD600 of 0.2-0.4 with 250 μM IPTG and was carried out separately for each expression strain. Post-induction expression was carried out at 30° C. under aerobic conditions for 30 minutes followed by 2-3 hours of anaerobic conditions for each expression strain separately. Afterwards, cells from both expression strains were harvested, concentrated, and re-suspended in 0.5 ml volume at OD600 of ˜40. For Example 15A, equal number cells from both strains were re-suspended in media containing 5-20 g/L glucose, 2.5-5 g/L glycerol, and 15 g/L potassium phosphate buffer (pH 7.2). After incubation for 24 hours at room temperature, the cells were centrifuged, and supernatant was filtered and analyzed via HPLC. For Example 15B, cells from expression strain 1 was suspended in media containing 5-20 g/L glucose, 2.5-5 g/L glycerol, and 15 g/L potassium phosphate buffer (pH 7.2). After incubation for 24 hours at room temperature, the cells were centrifuged, and supernatant was filtered and mixed with cells from expression strain 2. After incubation for 24 hours at room temperature, the cells were centrifuged, and supernatant was filtered and analyzed by HPLC.

(iii) HPLC analysis of HDO production: This was carried out as mentioned before. The results showed production of 400-800 mg/L of HDO.

Example 16: Synthesis of 3-hydroxy-propanal from glycerol

3-Hydroxy-propanal is synthesized from glycerol using glycerol dehydratases. Glycerol dehydratases can catalyze the dehydration in a coenzyme B12-dependent or coenzyme B12-independent manner in the presence of a reactivator protein. Coenzyme B12-dependent dehydratase is composed of three subunits: the large or “α” subunit, the medium or “β” subunit, and the small or “γ” subunit. These subunits assemble in an α2β2γ2 structure to form the apoenzyme. Coenzyme B12 (the active cofactor species) binds to the apoenzyme to form the catalytically active holoenzyme. Coenzyme B12 is required for catalytic activity as it is involved in the radical mechanism by which catalysis occurs. Biochemically, both coenzyme B12-dependent glycerol and coenzyme B12-dependent diol dehydratases are known to be subject to mechanism-based suicide inactivation by glycerol and other substrates (Daniel et al., FEMS Microbiology Reviews 22:553-566 (1999); Seifert, et al., Eur. J. Biochem. 268:2369-2378 (2001)). Inactivation can be overcome by relying on dehydratase reactivation factors to restore dehydratase activity (Toraya and Mori (J. Biol. Chem. 274:3372 (1999); and Tobimatsu et al. (J. Bacteria 181:4110 (1999)). Both the dehydratase reactivation and the coenzyme B12 regeneration processes require ATP. Shown below are a few examples of glycerol dehydratases, diol dehydratases and reactivating factors. One skilled in the art will recognize that glycerol dehydratases of Citrobacter freundii, Lactococcus reuteri, Clostridium pasteurianum, Clostridium butyricum, K. pneumoniae or their strains; diol dehydratase of Salmonella typhimurium, Klebsiella oxytoca or K. pneumoniae; and other dehydratase enzymes belonging to E.C. groups listed in Table 25 below or homologous enzymes of these sequences can also be used to carry out this step. Mutants of these enzymes (U.S. Pat. Nos. 8,445,659 & 7,410,754) can also be used herein to increase the efficiency of the process. In particular, coenzyme B12-independent-dehydratases (Raynaud, C., et al., Proc. Natl. Acad. Sci. U.S.A. 100, 5010-5015 (2003)) are favored for the industrial process due to the high cost of vitamin-B12.

TABLE 25 Exemplary biosynthesis polypeptides. Genbank ID EC Number Name Organism BAA08099.1 4.2.1.28 Diol dehydrase alpha subunit Klebsiella oxytoca BAA08100.1 4.2.1.28 Diol dehydrase beta subunit Klebsiella oxytoca BAA08101.1 4.2.1.28 Diol dehydrase gamma subunit Klebsiella oxytoca ABR24274.1 4.2.1.30 Glycerol dehydratase large subunit Klebsiella pneumoniae ABR24275.1 4.2.1.30 Glycerol dehydratase medium subunit Klebsiella pneumoniae ABR24276.1 4.2.1.30 Glycerol dehydratase small subunit Klebsiella pneumoniae AAM54728.1 4.2.1.30 Glycerol dehydratase Clostridium butyricum AAM54729.1 - glycerol dehydratase activator Clostridium butyricum ACI39932.1 4.2.1.30 B12-independent glycerol dehydratase Clostridium diolis ACI39933.1 - glycerol dehydratase activator Clostridium diolis ABQ83986.1 4.2.1.30 Glyerol dehydratase (B12-dependent) large Lactococcus reuteri subunit ABQ83985.1 4.2.1.30 Glyerol dehydratase (B12-dependent) Lactococcus reuteri medium subunit ABQ83984.1 4.2.1.30 Glyerol dehydratase (B12-dependent) small Lactococcus reuteri subunit ABQ83983.1 - Glyerol dehydratase (B12-dependent) Lactococcus reuteri activator large subunit ABQ83982.1 - Glyerol dehydratase (B12-dependent) Lactococcus reuteri activator small subunit

Example 17: Synthesis of Pyruvate

Conversion of Sugars to Pyruvate.

Conversion of sugars to pyruvate through glycolysis is very well known. In glycolysis, each mole of glucose gives 2 moles of ATP, 2 moles of reducing equivalents in the form of NAD(P)H and 2 moles of pyruvate.

Conversion of Glycerol to Pyruvate.

Glycerol can be converted to glycolysis intermediates both anaerobically and micro-aerobically. Anaerobically, glycerol is dehydrogenated to dihydroxyacetone which, after phosphorylation (using phosphoenol pyruvate or ATP), is converted to dihydroxyacetone phosphate a glycolytic pathway intermediate (Dharmadi, et al., Biotechnol. Bioeng. 94:821-829 (2006)). The respiratory pathway for glycerol conversion involves phosphorylation (by ATP) of glycerol followed by oxidation (quinone as electron acceptors) to give dihydroxyacetone phosphate that can be converted to pyruvate via glycolysis (Booth I R. Glycerol and methylglyoxal metabolism. Neidhardt F C, et al., editors. In: Escherichia coli and Salmonella: Cellular and molecular biology (web edition). 2005, Washington, D.C., ASM Press; Durnin et al., Biotechnol Bioeng. 103(1):148-161 (2009)).

Example 18: Preparation and Use of Microbial Organism for Production of 2,6-dihydroxy-hexanoate from 6-hydroxy-2-keto-hexanoate Intermediate

In some embodiments, the present disclosure provides technologies for producing 2,6-dihydroxy-hexanoate from 6-hydroxy-2-keto-hexanoate. Certain examples are described below.

Shown in FIG. 4 is a biosynthetic pathway for the production of 2,6-dihydroxy-hexanoate (6H2HH) from 6-hydroxy-2-keto-hexanoate intermediate. Shown below are examples incorporating the use of different 2-keto reductase enzymes for reduction of 6H2KH to 6H2HH i.e. 6-hydroxy-2-oxohexanoate 2-reductase. Examples of genes and corresponding enzymes from which they are encoded that were used to this step are shown in Table 26. Each enzyme therein may be substituted with homologous enzymes that belong to the same E.C. class.

(i) Preparation of Plasmids for 6H2HH Production:

The gene encoding 6-hydroxy-2-oxohexanoate 2-reductase was cloned on a plasmid with expression driven by T7 promoter using standard molecular biology methods. Escherichia coli was used as a target organism to engineer the 6H2HH production. The expression strains were obtained after co-transforming all three plasmids in electro competent E. coli BL21*(DE3) Δldh.

TABLE 26 Exemplary biosynthesis polypeptides. Uniprot ID or Genbank ID of 6-hydroxy-2- Annotated EC oxohexanoate 6H2HH Name Name Number 2-reductases Produced 1 D-2-hydroxyacid 1.1.99.6 WP_003431407.1 Yes dehydrogenase 2 ketopantoate 1.1.1.169 BAL51292.1 Yes reductase 3 2-ketogluconate 1.1.1.215 Q5FTU6 Yes reductase 4 D-lactate 1.1.1.28 AKC64094.1 Yes dehydrogenase 5 D-2-hydroxyacid 1.1.99.6 WP_002876862.1 Yes dehydrogenase 6 D-lactate 1.1.1.28 AGP69017.1 Yes dehydrogenase 7 D-2-hydroxyacid 1.1.99.6 WP_003640741.1 Yes dehydrogenase 8 phenyllactate 1.1.1.110 AKC64095.1 Yes dehydrogenase 9 D-lactate 1.1.1.28 AKC64094.1 Yes dehydrogenase

(ii) Cell Culturing, Protein Expression, and 6H2HH Production Analysis:

Starter cultures were grown overnight in tubes containing 10 mL LB media with appropriate antibiotics. Cell cultures for the expression and 6H2HH production were carried out in 100 mL volume using glass bottles. Complex growth medium was used and supplemented with 2 g/L D-glucose, 0.5 g/L potassium phosphate buffer (pH 7.2), and other substrates/nutrients important for enzyme expression. Pre-induction growth was carried out for ˜2 hours under aerobic conditions and at 30° C. Recombinant protein expression was induced at an OD600 of 0.2-0.4 with 250 μM IPTG. Post-induction expression was carried out at 30° C. under aerobic conditions for 60-90 minutes followed by 2-3 hours of anaerobic conditions. Afterwards, cells were harvested, concentrated, and re-suspended in 0.5 ml volume at OD600 of ˜40 in fresh medium containing ˜10 g/L glucose, 6-hydroxy-2-keto-hexanoate (5-10 g/L), and 15 g/L potassium phosphate buffer (pH 7.2). After incubation for 24 hours at room temperature, the cells were centrifuged, and supernatant was filtered and analyzed via HPLC.

(iii) HPLC analysis of 6H2HH production: Isocratic HPLC was used to detect and quantify 6H2HH. The method employed a Bio-Rad Aminex HPX-87 column, 0.7 mL/min of 0.5% formic acid (or 5 mM sulfuric acid) at 35° C. Detection was carried out using an RID (refractive index detector). The results showed production of 6H2HH from all strains of Examples 1-9 of Table 26.

Example 19: Preparation and Use of Microbial Organism for Production of 2,6-dihydroxy-hexanoate from Different Carbon Sources Via 6-hydroxy-2-keto-hexanoate Intermediate

In some embodiments, the present disclosure provides technologies for producing 2,6-dihydroxy-hexanoate from various carbon sources. Certain examples are described below. In some embodiments, the present disclosure provides technologies for producing 2,6-dihydroxy-hexanoate from pyruvate and 3HPA. In some embodiments, a yield is about or at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 mg/L, or is about or at least about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.7, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 250, or 300 g/L.

A biosynthetic pathway for the production of 6H2HH from pyruvate and 3-hydroxy-propanal through the 6-hydroxy-2-keto-hexanoate intermediate is shown in FIG. 4. Shown below are examples incorporating the use of aldolase-hydratase based two enzyme system for production of 6H2HH via this pathway. A glycerol dehydratase enzyme that is vitamin B12-independent or glycerol dehydratase enzyme that is a B12-dependent enzyme can be cloned to enable production of 3-hydroxy-propionaldehyde—a 6H2HH pathway precursor that can be made from glycerol using this enzyme. Although both types of glycerol dehydratases can be used herein, example shown herein uses the B12-dependent glycerol dehydratase enzyme. Each enzyme therein may be substituted with homologous enzymes that belong to the same E.C. class to yield 6H2HH.

(i) Preparation of plasmids & strains for 6H2HH production: MG1655(DE3) Δrne131, ΔldhA, Δ[frdB, frdC], ΔadhE, ΔpoxB, ΔpflB, Δ[ackA, pta] was used as the strain with the following plasmid comninations: Plasmid 1 (COLA replicon, kanamycin marker): Gene 1 (Glycerol dehydratase—pduCDEGH). Plasmid 2 (ColE1 replicon, ampicillin marker): Gene 2 (Ads-Hyd 8), Gene 2 (Qor-1), and Gene 3 (6-hydroxy-2-oxohexanoate 2-reductase—Q5FTU6).

(ii) Cell Culturing, Protein Expression, and 6H2HH Production Analysis:

Cell culturing (with appropriate antiobiotics), and protein expression was similar to that described in Example 1 for 3-hydroxy propanal. After incubation for 24 hours at room temperature, the cells were centrifuged, and supernatant was filtered and analyzed via HPLC.

(iii) HPLC analysis of 6H2HH production: Analysis was carried as our as mentioned in example 18. The strain was able to produce >1 g/L of 6H2HH under these conditions.

EQUIVALENTS

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this present disclosure belongs. All nucleotide sequences provided herein are presented in the 5′ to 3′ direction.

The embodiments illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising”, “including,” containing”, etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed.

It is to be understood that while the present technology has been described in conjunction with the above aspects, that the foregoing description and examples are intended to illustrate and not limit the scope of the present technology. Other aspects, advantages and modifications within the scope of the present technology will be apparent to those skilled in the art to which the present technology pertains.

REFERENCES

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Certain Sequences Uniprot or Sequence ID Genbank ID Number Sequence Information D7C0E5 SEQ ID MKGYTVPLSPRGIANLAPAPPWHYAGTVVGVEFFTDPAAAAATLPEGLTPDPDSAGRGVAMFIDWQY NO: 1 SSTGLEYLDPARSQYREFLITLDAHCNGAPVAWCPYIYVDNDAAMARGWVQGFPKKLGAVHQTRAY SVGGPGTPVLGPGGQFGATASSAGQRIAEAKITLEQPVPDPAALMSRPVINLRHFPRLAAGQHDQPAV HELVMSVLDDTAVSDAWVGTADLAFLPAHGEELADLPVRRTGKGFHFDLAYTVTDLMTLADHSA P0A144 SEQ ID MSNKIMKTSRLTAEDINGAWTIMPTPSTPDASDWRSTATVDLEETARIVEELIAAGVNGILSMGTFGEC NO: 2 ATLTWDEKRDYVSTIVETIRGRVPYFCGTTALNTREVIRQTRELIDIGANGTMLGVPMWVKMDLPTA VQFYRDVADAVPEAAIAIYANPEAFKFDFPRPFWAEMSKIPQVVTAKYLGIGMLDLDLRLAPNIRFLP HEDDYYAAARINPERITAFWSSGAMCGPATAIMLRDEVVRAKSTGDWAKAKAISDDMRAADSTLFPR GDFSEFSKYNIGLEKARMDAAGWLKAGPCRPPYNLVPEDYLAGAQKSGKAWAALHAKYSNELK Q79EM8 SEQ ID MTSPAVTSADITGLVGIVPTPSKPGSEAPDAVDTVDLDETARMVELIVASGVDVLLTNGTFGEVATLT NO: 3 YEELLAFNDTVIRTVANRIPVFCGASTLNTRDTIARSLALMGLGANGLFVGRPMWLPLDDEQLVSYYA AVCDAVPAAAVVVYDNTGVFKGKISSAAYAALAEIPQIVASKHLGVLSGSDAYASDLAAVKGRFPLL PTADNWLPSLEAFPGEVPAAWSGDVACGPEPVMALRRAIAEGLWDDARAVHEDIAWATEPLFPGGDI SKFMPYSIQIDRAEFEAAGYIVPGPSRHPYGTAPAAYLEGGAEVGRRWAGIRQKYVATLAEP A0A0N0AHI8 SEQ ID MKGYTYPLSPRGVANLAGKPPWHYVGDAVGVEFWTSPEAAAASLPTGLDPDPANPGHGYAVFIDWQ NO: 4 FNGATDDYLDPPFSQYSEFLVLLDAQWQGTPVAWCPFIWVDNDASLARGWVQGFPKKMGSIRQTRA FAIDSPAAPTVGKGGRFAAVMSAGGRRLAETTVTLDRTTDRLPALTRPLVNLRHFPRLSAGQHDNPA VHELTMSVLANLKFANTWIGTGELRFLPAPREELADLTPRRVGVGFRGSLSYTVNDLRIL A0A0N1FRY3 SEQ ID MKGYTVPLSPRGVANLAPAPPWHYAGTVVGVEFFTDPAAAAAALPEGLSSDPDSAGRGVAMFIDWQ NO: 5 YSSTDLEYLDPARSQYREFLVTLDAHYYGAPVAWCPYIYVDNDSAMARGWVQGFPKKLGAVHQTR AYSVGGQGTPVLGPGGQFGATASAAGQRIAEAKITLEQAVPDPAALMSRPVVNLRHFPRLTAGQHHK PAVHELVMSVLDGAAVSDAWAGTADLAFLPARGEELADLPIQRTGRGFHFDLAYTVTDLKTLIDHSN M3DYR1 SEQ ID MLKGYTVPLSPKGEANIAPTPPWHYAGDIVGVEFFTEPAAAEATLPEGLDPDPDTSGRVVAFFVDWQF NO: 6 NGERDEYLDPVRSQYREFFVLVDARHQGRPVSWCPYIYVDNHHALARGWIQGFPKKAGNVHQTRVF ASPGKASPTLSPGARFGASVSSDERTLAEARVTLEAPMEDPSALLSRDTINLRHFPTLEAGRYDKPAVH ELVRMDYADQQVADVWTGTSEITLFPAVGEELADLAPVRSGMGFRASMSYNVTQVEPLL W7SU48 SEQ ID MLGYSLPLSANGTANVVPAPPWHYAGDVVGVEFWTTPAAAAATLPSGLTPDPTTSGHAYALFVDWQ NO: 7 WAGSHQEYLDPVRSQYSEFLILMDAQFQGRAVAWCPYIWVDNDAALARGWFQGFPKKLGAIRQTRA FSVPGQASPVVGPGGQFGASLSAAGRRLAEAQITLQAPSATLPALGRPIVNLRHFPRLIAGQYDNPSVH ELTQSVLDTPVVGNNWTGTSTLNFFTAPGEELADLQPVRTGSGFRGSLSYTVTTLKMLSGPDA A0A286PH18 SEQ ID MKGYTVPLSPRGIANLAPAPPWHYAGTVVGVEFFTDPAAAAATLPEGLTPDPDSAGRGVAMFIDWQY NO: 8 SSTGLEYLDPARSQYREFLLTLDAHYNGTPVAWCPYIYVDNDSAMARGWVQGFPKKLGAVHQTRAY SVGGPGTPVLGPGGQFGATASAAGQRIAEAKVTLEQPVPDPAALMSRPVVNLRHFPRLAAGQHDKPA VHELVMSVLDGVAVSDAWAGTADLAFLPAHGEELADLPVQRTGRGFHFDLAYTVTDLKTLIDRSN Q9X9Q6 SEQ ID MARTLMKPDDVKGAWAIIPTPAKDDASDWRATKTVDLDETARVVNGLIDAGINGILSMGTLGEAAT NO: 9 MTHDEKLDFIKALVDAAAGRVPIFVGTTCLNTRDTIALTRQALDIGADGTMLGVPMWCAPSVDVAVQ FYKDLAEAVPEMNIAIYANPEAFKFDFPRSFWAQVAEIPQVVTAKYIGVAHLLPDLAAIRGRIKLLPIDF DYYGAARMDESIDAFWSSGAVCDPLVTTTLRDLVSQARATGDWSAARAFMGRLGPTAAPLFPNGSF KEFSTYNIALEKARMNAGGWMNAGPVRPPYHLCPEPYLEGARLSGRMWAELGKALAAEK Q9WXH7 SEQ ID MAKSGLLNASDIHGVWSILPTPSKPDASDWRATNTVDLDETARAVEGLIAAGANGILSMGTLGECESL NO: 10 TWEEKKVFMQTIVETARGRVPVFVGTTTLNTRDTIEQTRYAHSIGADGTMLGIPMWCNPCVDMAVQ YYKDVAEAVPEMNIAIYANTEAFKFDFPRAFWARVSEIRQVVAAKYIGIEFLLQDLHLTKHRMKLLPL DYQYYAAARMDDFVDAFWSSGTVCGPLVSTTLRDKVIAARRTKDWTDAHAFQGRLVKTAAPFPEDS FKTFSIYNVALEKGRIDAAGWMNAGPVRPPYNDICPASYLDSWKASGQRWAELHKQLETESSGK A4XDS1 SEQ ID MARELLTAADVKGAWAIVPTPAKEGASDWRAADTVNVEEAARMIDGLIEAGVDGILSMGTLGEAAT NO: 11 MTLDEKLVFMKTIVDTAAGRVPVFVGTTCINTRDTIALTRKAVDIGATGTMLGVPMWCAPSVDVAV QFYRDVAEAVPDINIAIYANPEAFKFDFPRTFWGQVAEIPQVVTAKYIGVGTLLPDLAAIKGRIKLLPID FDYYGAARMDDSIDAFWTSGAVCHPLVSTTLRDVVAAARASGDWSAAKAFMGRLAPTAATLFPNGS FKEFSTYNIPLEKARMTAGGWMNAGPCRPPYHLCPENYLEGARNSGRMWAELGKALEAER F2J6N9 SEQ ID MTRKLLTVDDVNGCWAIMPTPSKPGASDPNAVDTVDLEETARAAEALVAAGVDGILSLGTFGEAATT NO: 12 TWEEKQAFMRTLVETVRGRVPVFGGTTSLNTRDTIRMTRAAREIGVDGVMLGLPMWVQPDLATAVQ FFRDVASACPDVAICAYANPEAFKFEFPRAFWAQIADIPQIVSAKYIHTAGLYADLNLTKRRIRLMPLD VDYYAAARIDPDACTAFWTSGAVCGPAPAIQLRDLVSKAKKTGDWTGAKKLTDRIGQTYRTLFPNGS FKDFSVYNIGIEKARMDAAGWMKAGPCRAPYSLVPEPYLEGARESGRQWAKLAAELATERAE A0A063BFL5 SEQ ID MIHPKLRIDASGINGLWPILPTPAKPNASDWRERSTVDLDETARIVESLIDAGVDGLLSLGTYGEAHSL NO: 13 LWEEKKAFVGCVLETIRGRIPFFTGTTALNTREVVEQTRAMHDMGVSGTMLGVPMWCKTDLATAVQ FFRDVTEACPDTALAIYANTEAFKFEFPRPFWAEIGKMPQAVACKYLGIGMLAVDLELAPNMRFLPNE QDYYAAARIDPERVTAFWSSGALCGPLPALTLRDRVARAKSSNDWTSAKEIADRMRACDVGFFPKGE FSEFSKFNAPLEKARMNTAGYVNAGPCRPPYHVIPQEYLAGAERSGRAHAALNAELKQAEHSI Q9ZHH6 SEQ ID MSKQRKQRLGTEDVNGAWVIMPTPAKPEASDWRATDTVDLDETARIVEALIDSGVNGILSLGTFGEC NO: 14 ATLTWEEKQAFIGAVVETTRGRVPFFCGTTALNTREVVRQTRAALDIGVDGTMLGVPMWSRMEVPA AVQFYRDVAEACPEAAIAVYANADAFKFEFPRAFWAQVAQIPQVVTAKYLGIGMLDLDLTLAPGIRF LPHEDDYYAAARVAPERVTAFWSSGAMCGPATAIRLRDEVAKAKQTGDWRLAKELSDAMRRADAT LFPRGDFAEFSKYNIAIEKERMNAAGWLRAGPCRPPYHIAPEEYLDGARQSGRAWAELHQQYSDL A0A0C1K853 SEQ ID MMSDMVKPRMTADDVNGVWVIMPTPAKPDASDWRVENTVDLDETVRIVENLLASGVNGIMSNGTF NO: 15 GECATLTWDEKRDFIATVAETIKGRVPFFCGTTALHTREVIRQTREVMRLGADGVMLGLPMWCKME TPSAIQFYRDVAEAVPDAAIAVYANPEAFKYEFPREFWAQVSEIPQVVTAKYLGIGMLDLDLRLASSIR FLPHEDDYYAAARINPERMTAFWSSAAMCGPATPLKLRDAVADAKVTGKWSVAKAISDEMRKADS MLFPKGDFSEFSKYNIGLEKARMDEAGWLKAGPCRPPYHVIPEMYLEGARKSGRAWAELHAKYSAE G WP_ SEQ ID MAKQKKSRMTAEDIHGAWVIMPTPATPDASDWRVQHTVDLEETARIVEALIAAGVNGIFSNGTFGEC 034398482 NO: 16 ATLTWEEKRDFIATVVETARGRVPFFCGTTALHTREVIRQTREAMDIGASGTMLGVPMWCKMEVPTA VQFYRDVAEAVPEAAIAIYANPEAFKFDFPRSFWAQVSNIPQVITAKYLGIGMLDLDLRLAPSIRFLPHE DDYYAAARIDPERMTAFWSSGAMCGPATAIRLRDTVGAAKRSGDWTDAKAISDAMRQADSTLFPRG DFSEFSKFNIGLEKARMDAAGWLKAGPCRPPYHIVPEEHLAGARKSGEAWAALHARYATLD PYK12191 SEQ ID MNTAKLIGFNYPLTPKGKSTLNPPPPWYYSSDFLDVEFWAQPAAVASLLPNGLEPDPAANGHCNALF NO: 17 YDWQFSGDNEEYLDPARYQYREFFILVDALFEGRSVSYCPYIFVDNDAALARGWTQGYPKRLGQVFQ TRYYAATSKAGPALAPGSKFAGSLTAAGQLIAEAVVTLRQAVTDPSLLKQKPVINLLHVPRLAADKH DKPAIHELVENVPSSVKIEQAWIGEGSLTLPVCRGEEISDLAPLRCGKGIRASMAYVVDDLKTLKDLRN A0A370X7D8 SEQ ID MKSNFFVPMTPRGLSNISPPPPWHYAGDFLIIDFWARPDAVASLLPAELQPDVKAEGHAQAYFIDWQY NO: 18 TAAHDEFLDPARYQYREFFVLVDALFQGKPVAFCPYIFVDNDAAIARGWAQGFPKRYGTILQTRLFAA SGPASPKLAPGGRFGASASTAGQRIARGLVTLEKAVTDPAALGSRPTINLRHFPRLAAGQWERPAVHE LVESVMDNFTVADAWMGKGELTLPECENEELSDLAPVRCGNGYRMSVSYSVTDLKTLVDHSAK WP_ SEQ ID MLKGYMAPLSPLGKASINPPPPWHYSGDVIGAEFWAEPEATAATLPPGLDPDPSTAGHGVVLFIDWQF 028222253 NO: 19 TAQDDEFLDPARYQYRECLFLVDAVHKGTPVMWCPYIYVDNDAALARGWAQGFPKKLASVYQTRT FAAPSAAAAPVASGSRFGASLSAHGERLAEARITLRQPVADPKSLLARPTVNRRYFASLVAGLHDKPA VDELVLSVTDNLSVADAWAGDAELLFPDARGEEICAFGPVKVGGGFRFSLAYSVTDLKLLEDLTRLG K F2J6L6 SEQ ID MKRDMLTVDDVTGCWAIMPTPSKPNASDPSATDTVDLDETARVAEALVAAGVDGILSLGTLGECAT NO: 20 TTWDEKQAYMRTLVETLRGRIPVFGGTTGLNTRDSIAMTRAAREIGVDGVMLGLPMWVQPDVPTAV QFYRDVAAACPDVAICVYANPEAFKFEFPRAFWAQIAEIPQVVSAKYINIAALYTDLNLTRRRIRLMPL DVDYYAAARVDPEACSAFWTSGAVCGPAPAIQLRDLVLEARQSGDWSKAKALTDRIGMTYRTLFPN GSFKEFSVYNIGIEKARMDAAGWMTAGPVRPPYHIVPEAILEGGRESGRQWAKLAAELEREAGR A0A0N0L9F6 SEQ ID MTQSYTTPLTPRGLSSIAPPPPWHYSGDFLVVEFWADPIAVANTLPAGLTVDSASPGHASAVFVDWQF NO: 21 TGENDELLDPARYQYREFFILLDALHEGQPVSYCPYIFVDNDSALMRGLIQGFPKRLGAVHQTRTFSAP SRAAAQVEPGARFAATASTAGQRIARGEVQLQHKIDDVSKLGFGARPLINLRHFPRLATGQHNDPAV HELVVSVMDNPNIVDAWAGEGNLVFPQAEGEEVSDLAPTRVGAGFRASMSYTVTDLKALPNATIER A0A1G9YWG7 SEQ ID MLRGFTVPKSPFGQAALTPPPPWHYAGDVVGVEFWTDPEATAATLPNGLSPDPNSNGHAVMMFLDW NO: 22 QFTAQDDEYLEPARYQYREAFILVDAMYRDEPVMWCPYIYVDNDAALARGWTQGFPKKMGSIFQTR SFAASGPAAAPVASGSRFGASLSAHGQRLAEACVTLHRPVENGLSLLSRPTVLLRYFPRLAAGYQDKP AVNELAMSITDNLTVAGAWIGKGELNFPEASGEELNALAPKRIESGFRYSLSYSVSDLKILEDHGSQ A0A2U1BT09 SEQ ID MSTKRTLMTANDVQGAWAIMPTSAKDGSESWRMTDSLDLDATVAAINGLIDSGVDGILTMGTYGEA NO: 23 ATLTVDEKKRFMACLVETVAGRVPCFVGTTTLNTRDTIELTRYAADLGADGTMLGLPMWCAPTLPA AVRFYRDVAEACPDMAQCIYANPEAFRFDFPPPFWAQVADIPQVVSAKFTSVGHLIQNLEITRGKVRA LPIELDYYAATRVDDDVCAFWSSGAVCGPTPTIALRDEITRAKTSGDWTKAKELTDKMWAAVTPMFP AGGFREFSMYNIAIDKMRMQTAGWMRVGPTRPPYDMMPDHIRGGAVEAGKLWAELAKATVLAGA A0A244DHE8 SEQ ID MSKQYAVPLSPRGLSSIAPPPPWHYSGDFLIVEFWADPAAVAATLPAGLSVDPSSPGHATALFVDWQF NO: 24 TGQNDELLDPARYQYREFFLLVDALYEGQPVAYCPYIFVDNDSAMMRGLIQGFPKRLGAVHQTRTFA APSLAAAQVAPGARFAATASTAGQRIARAEVKLTGKVDDPSTVSLAGRPIVNLRHFPRLAAGQHETPA VHELVMSIMDDPRMADVWAGEGQLSLPVAEGEEISDLAPVRVGAGYRLSMSYTVTDLKTLSDGTQA A WP_ SEQ ID MKKPLLTVDDVTGCWAIMPTPSKPNGSDINATDTVDLDETARAAEALVASGVNGILSQGTFGEAATT 107818191 NO: 25 TWEEKQAFLRTLVETVDGRVPVFGGTTSLNTRDTIRMTKAVREIGVDGVMLGPPMWCQPDVPTAVQ FFRDVAEACPDTAICAYANPEAFKFDFPRAFWAQIAEIPQVVSAKYMNIAALYMDLNLTGRKIRLMPL DMDYYAAARMDPEACTAFWTSGAICGPEPVIQLRDLVAEAHKTGDWGKAKALTDRIAATYRTLFPN GSFKEFSVYNIGIEKARIDAAGWMTAGPCRPPYHVIPEPILDGAREAGLQWAKLVSALESEKTA A0A023WZF9 SEQ ID MSNKTMKPARLTAEDIHGVWAIMPTPATPDASNWRSTNTVDLNETARIVEELIAAGVNGILSMGTFGE NO: 26 CATLTWEEKRDYVSTIVETIRGRVPYFCGTTALNTREVIRQTREFMDMGASGTMLGVPMWVKMDLP TAVQFYRDVAEAVPEAAIAIYANPEAFKFDFPRPFWAEMSKIPQVVTAKYLGIGMLDLDLKLAPNIRF LPHEDDYYAAARINPERMTAFWSSGSMCGPATAIMLRDAVDQAKSSGDWIKAKAISDDMRAADSTL FPRGDFSEFSKYNIGLEKARMDAAGWLTAGPCRPPYNIVPEDYIAGALKSGKAWAALHAKYSKELK PYN48855 SEQ ID MLKGFNYPLTPKGKSTLNPSPPWHYSADFLDIEFWSEPSAVTAVLPAGLDPDPAANGHGHALFYDWQ NO: 27 FAGENEEYLDPARYQYREFFLLVDALYEGQPISYCPYIFVDNDAAIARGWTQGYPKRLGQVFQTRYY AATGKAGPALAPGSKFAGSLTAGGQRLAEALVTLKEPVTDPALLKQRPIVNLLHYPQLAADKQDEPAI HQLVENVPHDLKIEQAWIGDGSLTLPVCRSEELSDLAPVRCGKGIRASMAYIVDDLKTLKDLTKGFSL LA A0A421PAQ6 SEQ ID MLKGYTVPLSPKGEANIAPTPPWHYAGDIVGVEFFTEPSAAEATLPEGLDPDPDTSGRVVAFFVDWQF NO: 28 NGEQDEYLDPVRSQYREFFVLVDARHQGRPVSWCPYIYVDNHHALARGWIQGFPKKAGNVHQTRVF ASPGKASPTLSPGARFGATVSSDERTLAEARVTLEAPMEDPSALLARDTINLRHFPTLEVGKYDKPAV HELVRMDYADQQVADVWTGTSEITLFPAVGEELADLAPVRPGMGFRASMSYNVTQVEPLG WP_ SEQ ID MNKPYAVPLSPRGLSSIAPPPPWHYAGDFILVEFWADPAAAAAVLPKGLSLDPASPGHATALFIDWQF 028217297 NO: 29 TGSNDEMLDPARYQYREFFVLVDALHEGKPVSFCPYIFVDNDSAMMRGLIQGFPKRYGQIHQTRTFA ALSPAAAPVTAGTRFAATASAAGQRLAHAEVKLEAAVQDVSKLGIAGRPVVNQRYFPRLAAGQHDT PAVNELVLSIMDNAQIADVWAGEGKLTFPFAQGEEIADLQPVRVGAGFRGSMAYSVTDLKTLVDHTK WP_ SEQ ID MLKGFTLPKSPFGQAALTPPPPWHYSGDVIGVEFRTDPSATAATLPNGLSPDPKSNGHAVMMFVDWQ 034507049 NO: 30 FTAQNDEYLDPARYRYREAFVLLDAVYRNAPVMWCPYVFVDNDAALARGWTQGFPKKIGSIFQTRT YAAASPAAAPVAPGGRFGASLSAHGQRLAEARITLQEPVEDGLSLLSRPTVLLRYFPRLAAGYQDKPA VNELTMAITDNLTVADAWIGDGELNLPEVHGEELHGLAPIAIESGFRYSLSYSVTDLKILEDHAS Q47098 SEQ ID MENSFKAALKAGRPQIGLWLGLSSSYSAELLAGAGFDWLLIDGEHAPNNVQTVLTQLQAIAPYPSQPV NO: 31 VRPSWNDPVQIKQLLDVGTQTLLVPMVQNADEAREAVRATRYPPAGIRGVGSALARASRWNRIPDYL QKANDQMCVLVQIETREAMKNLPQILDVEGVDGVFIGPADLSADMGYAGNPQHPEVQAAIEQAIVQI RESGKAPGILIANEQLAKRYLELGALFVAVGVDTTLLARAAEALAARFGAQATAVKPGVY P75682 SEQ ID MPQSALFTGIIPPVSTIFTADGQLDKPGTAALIDDLIKAGVDGLFFLGSGGEFSQLGAEERKAIARFAIDH NO: 32 VDRRVPVLIGTGGTNARETIELSQHAQQAGADGIVVINPYYWKVSEANLIRYFEQVADSVTLPVMLYN FPALTGQDLTPALVKTLADSRSNIIGIKDTIDSVAHLRSMIHTVKGAHPHFTVLCGYDDHLFNTLLLGG DGAISASGNFAPQVSVNLLKAWRDGDVAKAAGYHQTLLQIPQMYQLDTPFVNVIKEAIVLCGRPVST HVLPPASPLDEPRKAQLKTLLQQLKLC P0A6L4 SEQ ID MATNLRGVMAALLTPFDQQQALDKASLRRLVQFNIQQGIDGLYVGGSTGEAFVQSLSEREQVLEIVA NO: 33 EEAKGKIKLIAHVGCVSTAESQQLAASAKRYGFDAVSAVTPFYYPFSFEEHCDHYRAIIDSADGLPMV VYNIPALSGVKLTLDQINTLVTLPGVGALKQTSGDLYQMEQIRREHPDLVLYNGYDEIFASGLLAGAD GGIGSTYNIMGWRYQGIVKALKEGDIQTAQKLQTECNKVIDLLIKTGVFRGLKTVLHYMDVVSVPLC RKPFGPVDEKYLPELKALAQQLMQERG P23522 SEQ ID MNNDVFPNKFKAALAAKQVQIGCWSALSNPISTEVLGLAGFDWLVLDGEHAPNDISTFIPQLMALKG NO: 34 SASAPVVRVPTNEPVIIKRLLDIGFYNFLIPFVETKEEAELAVASTRYPPEGIRGVSVSHRANMFGTVAD YFAQSNKNITILVQIESQQGVDNVDAIAATEGVDGIFVGPSDLAAALGHLGNASHPDVQKAIQHIFNRA SAHGKPSGILAPVEADARRYLEWGATFVAVGSDLGVFRSATQKLADTFKK P0A955 SEQ ID MKNWKTSAESILTTGPVVPVIVVKKLEHAVPMAKALVAGGVRVLEVTLRTECAVDAIRAIAKEVPEAI NO: 35 VGAGTVLNPQQLAEVTEAGAQFAISPGLTEPLLKAATEGTIPLIPGISTVSELMLGMDYGLKEFKFFPAE ANGGVKALQAIAGPFSQVRFCPTGGISPANYRDYLALKSVLCIGGSWLVPADALEAGDYDRITKLARE AVEGAKL Q6BF16 SEQ ID MQWQTKLPLIAILRGITPDEALAHVGAVIDAGFDAVEIPLNSPQWEQSIPAIVDAYGDKALIGAGTVLK NO: 36 PEQVDALARMGCQLIVTPNIHSEVIRRAVGYGMTVCPGCATATEAFTALEAGAQALKIFPSSAFGPQYI KALKAVLPSDIAVFAVGGVTPENLAQWIDAGCAGAGLGSDLYRAGQSVERTAQQAAAFVKAYREAV Q M9YI86 SEQ ID MPAPVLAATSPGAGRAIHLINPAMPAFRAAFEETLMKMPHNAFKAALQRPETQYGIWAGFASGYAAE NO: 37 IVAGTGYDWMLIDGEHAPNSVPTILAQLQSVAPYPTQPVVRPVCGDPVLIKQLLDIGAQTLMVPMVES AEQARALVRAMRYPPHGIRGVGGGLARATRWDGVPDYLNTAHEELCLIVQVESRAGVENVEAIAAV EGVDAVFIGPADLSIGLGHPGDPGHPQVQELIHHAIEATRAAGKACGILAPHEEDARRYREWGCRFIA VAIDISLLRQGALAGLARFRDTPASDAPSRTY Q8NMD2 SEQ ID MASATFTGVIPPVMTPLHADGSVDVESLRKLVDHLINGGVDGLFALGSSGEAAFLTRAQRKLALTTIIE NO: 38 HTAGRVPVTAGVIETTTARVIELVEDALEAGAEGLVATAPFYTRTHDVEIEEHFRKIHAAAPELPLFAY NIPVSVHSNLNPVMLLTLAKDGVLAGTKDSSGNDGAIRSLIEARDDAGLTEQFKILTGSETTVDFAYLA GADGVVPGLGNVDPAAYAALAKLCLDGKWAEAAALQKRINHLFHIVFVGDTSHMSGSSAGLGGFKT ALAHLGIIESNAMAVPHQSLSDEETARIHAIVDEFLYTA A0A1J6QD42 SEQ ID MDKNIIIGAMTALITPFKNGKVDEQSYARLIKRQIENGIDAVVPVGTTGESATLTHEEHRTCIEIAVETC NO: 39 KETKVKVLAGAGSNATHEAVGLAKFAKEHGADGILSVAPYYNKPTQQGLYEHYKAIAQSVDIPVLLY NVPGRTGCEISTDTIIKLFRDCENIYGVKEASGNIDKCVDLLAHEPRMMLISGEDAINYPILSNGGKGVI SVTSNLLPDMISTLTHFALDENYKEAKKINDELYNINKILFCESNPIPIKTAMYIAGLIESLEFRLPLCPPS KENFAKIEEVMKKYKIKGF Q8RBI5 SEQ ID MPVFKGSCVAIVTPFTENGVNFDKLGELIEWHIKEGTDAILICGTTGEASTMTDEEQKEAIKFTVEKVA NO: 40 KRIPVIAGTGSNNTAHAIELSEYAQSVGADALLVITPYYNKTTQKGLVAHFTEIARHVDIPIIIYNVPSRT SLNMLPETYLEVKKKAENVVGVKEASGDISQIAEIARIMGKSFEIYSGNDDQVIPIMSLGGLGVISVTA NIIPAKIHEMTTAYLNGDIEKARDMQLELNPLNKALFIETNPIPVKTAMNLMGFGVGPLRLPLVEMSEK NLEYLKSVLRQYGLLKEEN A3LZU9 SEQ ID MTISAALPKRGVYTPVPTFFKKDLHTIDYDSQIEHAKFLQQNGITGLVLLGSTGENSHLTRKERIELVST NO: 41 IHEELPDFPLMAGVAQNSVEDAIEEILQLKNAGAQHALVLPSSYFGASIKQQGIIDWYTEVADNASLPV LIYVYPGVSNNISIDPRTIKKLSAHPNIVGAKISHGDVSHHAIIGLDQEIAANQFITLTGLGQILLPVLVVG IQGTVDALCGAFPKIYVKLLENYDKGDLRAAAELQLVISRAEELVVKFGVVGIKKAIHFATGIGETYLG RAPLTQDVNDADWKSYNDYLLGIVSVESTL Q4JC35 SEQ ID MEIISPIITPFDKQGKVNVDALKTHAKNLLEKGIDAIFVNGTTGLGPALSKDEKRQNLNALYDVTHKLI NO: 42 FQVGSLNLNDVMELVKFSNEMDILGVSSHSPYYFPRLPEKFLAKYYEEIARISSHSLYIYNYPAATGYDI PPSILKSLPVKGIKDTNQDLAHSLEYKLNLPGVKVYNGSNTLIYYSLLSLDGVVASFTNFIPEVIVKQRD LIKQGKLDDALRLQELINRLADILRKYGSISAIYVLVNEFQGYDVGYPRPPIFPLTDEEALSLKREIEPLK RKIQELVH O54288 SEQ ID MPEIITPIITPFTKDNRIDKEKLKIHAENLIRKGIDKLFVNGTTGLGPSLSPEEKLENLKAVYDVTNKIIFQ NO: 43 VGGLNLDDAIRLAKLSKDFDIVGIASYAPYYYPRMSEKHLVKYFKTLCEVSPHPVYLYNYPTATGKDI DAKVAKEIGCFTGVKDTIENIIHTLDYKRLNPNMLVYSGSDMLIATVASTGLDGNVAAGSNYLPEVTV TIKKLAMERKIDEALKLQFLHDEVIEASRIFGSLSSNYVLTKYFQGYDLGYPRPPIFPLDDEEERQLIKK VEGIRAKLVELKILKE F9VPG1 SEQ ID MDIVTPILTPFTKEGKIDVEKLKAHAKFLIDNGIDLLFVNGTTGLGPALSKEEKLTTLKTIYDVTNKVIF NO: 44 QVGSLNINDVIDLVKASKDFDIVGIASYPPFYFPRLPEKFLLKYFTTIANYSPHSLYIYNYPLATGYDISA KIVYQMKDLITGLKDTNQDLSHSLEYKILMPNLKVYNGSDSLVFYSLTSLDGSVTAASNYLPHVMKK MKEHITSGQVSKAIELQKLINKALDISRKYGQLSAIYYLVKEFLGYDVGYPRGPIFPLEEDEVKALLSEI QPVKKEIERAVS P28304 SEQ ID MATRIEFHKHGGPEVLQAVEFTPADPAENEIQVENKAIGINFIDTYIRSGLYPPPSLPSGLGTEAAGIVSK NO: 45 VGSGVKHIKAGDRVVYAQSALGAYSSVHNIIADKAAILPAAISFEQAAASFLKGLTVYYLLRKTYEIKP DEQFLFHAAAGGVGLIACQWAKALGAKLIGTVGTAQKAQSALKAGAWQVINYREEDLVERLKEITG GKKVRVVYDSVGRDTWERSLDCLQRRGLMVSFGNSSGAVTGVNLGILNQKGSLYVTRPSLQGYITTR EELTEASNELFSLIASGVIKVDVAEQQKYPLKDAQRAHEILESRATQGSSLLIP P40783 SEQ ID MATRIEFHKHGGPEVLQTVEFTPAEPAEHEIQVENKAIGINFIDTYIRSGLYPPPSLPAGLGTEAAGVVS NO: 46 KVGNGVEHIRVGDRVVYAQSTLGAYSSVHNVTADKAAILPDAISFEQAAASFLKGLTVFYLLRKTYE VKPDEPFLFHAAAGGVGLIACQWAKALGAKLIGTVGSAQKAQRALDAGAWQVINYREESIVERVKEI TGGKKVRVVYDSVGKDTWEASLDCLQRRGLMVSFGNASGPVTGVNLGILNQKGSLYATRPSLQGYI TTREELTEASNELFSLIASGVIKVDVAENQRYALKDARRAHEVLESRATQGSSLLIP Q0K2I0 SEQ ID MPRHGCLTIVTVAPMIAARAGHDNQETALAKAIRMYETGGPEVLRYEDAEVGDPGPGEVRIRHAAVG NO: 47 LNYADTYFRNGTYPVPLPGGMGVEAAGVVQAVGPGVTHVAEGDRVTYTGFINTLGAYSTERLVPAA PLIRLPEAISFETAAAMTMRGLTSAYLMRRIYPFQGGEAILLHAAAGGVGLIVSQWARLLGLTVIGTVS TEAKAEVARAHGCDHIINYSHEDVAKRVRELTDGAGVSVVFDSVGKSTFMASLDSLKRRGLMVCVG TASGTIPPFDPQLLARKGSVYLTRPALADYIADPAEKAELAAEVFGHVAAGRIRIEINQRYALQDAVQA HRDLESRKTTGSSIFVL A0A1Z1SRY9 SEQ ID MAKRIQFAAHGNADVLELTSFTPAPLGDNEVQVANKAIGINYIDTYVRSGLYPVEHFPSGLGTEAAGV NO: 48 VIKTGAHVTSLKEGDRVVYAQSPLGAYSDTHNVPENKVARLPDNISFEQAAASFLKGLTVYYLFNETY KLRAGETFLFHAAAGGVGLIASQWAKAIGAKMIGTAGSDEKVAKAKAAGAWKVINYQTESIVERVL ALTNNQKVPVVYDSVGKATWLDSLHCLQRRGLMVSFGNASGAVTGVDLGILNKLGSLYVTRPSISGY ITTREELDAASEALFTLIGRGKIDVSVPDNQKFALADAKAAHRYLESRQSQGSSLLIP P43903 SEQ ID MAKRIQFAAYGGPEVLEYRDYQPAEPGPREVRVRNRAIGLNFIDTYYRSGLYPAPGLPSGLGSEGAGE NO: 49 VEAVGSEVTRFKVGDRVAYATGPLGAYSELHVLAEEKLVHLPDGIDFEQAAAVMLKGLTTQYLLRQ TYELRGGETILFHAAAGGVGLFACQWAKALGVQLIGTVSSPEKARLARQHGAWETIDYSHENVARRV LELTDGKKCPVVYDSVGKDTWETSLDCVAPRGLLVSFGNASGPVTGVNLGILSQKGSLYVTRPTLGS YADTPEKLQAMADELFGLIERGDIRIEINQRFALAEAARAHTELAARRTTGSTVLLP I7G8G0 SEQ ID MHAIEVAETGGPEVLNYIERPEPSPGPGEVLIKADAIGVNFIDTYFRSGLYPRELPFVVGTEVCGTVAAI NO: 50 GNDVAALKVGDRVVTANAVGAYADYCVAPADFVAYVPDGVAPEAVASALLKGMTAHYLLKSTYP VQPSDTVLVHAGAGGVGLILTQWATSLGTRVITTASTPEKAELSRQAGAVEVLDYPDPDDPQPFASRV RELTGGAGVAAVYDGVGATTFDASLASLAVRGTLALFGASSGPVPPFDPQRLNAAGSVFLTRPTLAH HTRTADEFSWRAGELINAIADGSIKITVGGTYPLAEASRAHTDLQGRKTVGSIVLIP Q142L2 SEQ ID MVKAIRFDKTGGPEVMKWVDVEVGEPGAGEIRVRQTAVGLNYIDVYFRTGLYPLPLPGGLGMEAAG NO: 51 EVTALGSGVSGLKVGDRIAYVARPPGAYAQERVLQAAQVVKVPDALTDEQAASVMLQGLTAQYLLR RTYPVKAGDTILIQAAAGGVGLLVCQWAKALGATVIGTVGSDEKAEIATAHGCDHAIVYTRENFTRR VREITNGAGVPVVYDSIGKDTFTGSLDCLAPLGMFVSFGNASGPLPPIDSSEFAGRGSLFFTRPTLFTYI AKRSDYEAMSTELFDVLVSGKVKTSINQRYALADVGRAHADLEGRRTTGSTVLLP ALK19324.1 SEQ ID MPKAIRYDQPGGPDVMKWVDVEVGEPKAGEVRIRQHAVGLNYIDVYFRTGLYSQPLPGGLGMEAAG NO: 52 EVTAVGEGVTALKAGDRVAYVGQPPGAYAQERVMPAERLVKLPDGISYDDAASVMLQGLTAHYLL RRTYPVKAGDTILIHAAAGGVGLLVCQWAKALGATVIGTVGSDEKAALAKAHGCDHPIVYTRENFTQ RVKEITNGAGVPVVYDSIGKDTYIGSLDCLAPLGYFVSFGNASGPLPAIDSKEFSSRGSLFFTRPTLFSYI AKRADLESAAAELFDVILSGKVKTSINQRYPLAEVGRAHADLESRNTTGSTILVP Q5FTU6 SEQ ID MSSKPDILTIDPLVPVMKERLEKSFTLHPYTSLENLKNIAPAIRGITTGGGSGVPSEIMDALPNLEVISVN NO: 53 GVGTDRINLDEARRRNIGVAITQNTLTDDVADMAVALMMAVMRSIVTNDAFVRAGKWPSATAPLGR SLTRKKVGIAGFGHIGQAIAKRVSAFGMEVAYFNSHARPESTCHFEPDLKALATWCDVLILAVSGGPR SANMIDRDTLDALGKDGFLVNIARGTVVDEAALLSALQEKRIAGAGLDVFQNEPNINPAFLSLPNTVL QAHQASATVETRTTMANLVVDNLIAYFTDKTLLTPVI A0A1V9IP73 SEQ ID MKILAYCVRPDEIDSFKNFSEKYGHTVDLIPDSFGPSVAHLAKGYDGISILGNDTCNREALEKIKDCGIK NO: 54 YLATRTAGVNNIDFDAAKEFGINVANVPAYSPNSVSEFTVGLALSLTRKIPFALKRVELNNFALGGLIG VELRNLTLGVIGTGRIGLKVIEGFSGFGMKKMIGYDIFENEKAKEYIEYKSLDEVYKEADIITLHAPLTD DNYHMIGKESIAKMKDGVFIINAARGALIDSEALIEGLKSGKIAGAALDSYEYEQGVFHNNKMNEIMK DDTLARLKSFPNVVITPHLGFYTDEAVSNMVEITLMNLQEFELKGTCKNQRVCK T4VW93 SEQ ID MDNKALLKGVRVVELSSFVAAPCCAKLLGDWGAEVIKIEPLGGDGIRVMGGTFKSPCTDEENPMFEL NO: 55 ENGNKKGISVNVKTKEGVEIIHKLLAKADIFITNVREQALSKIGLTYDQLKDEFPALIHAHILGYGENGP LKDKPGFDYTAYFARGGVSQSLMEKGTSPCNTAAAFGDHYAGVSLTAGILAALYKKQMTGEGDRVT VSLYHTALYGMGMMITTAQYGNKMPISRANPNSPLMTTYKCKDGKWIQLALIQYNKWLPKFCNVIN RPEIMEDERFNDIKVMPLHVDEMVEIVGEAMLEKTLDEWSALLEEADLPFEKVQSCEDILEDEQAWA NDFLFKTKYANGNEGVLVNGPVKFKTMGIKEYTPAPRVGEHTEEVLKELGYTEEEILNMVNSQAVKL DDSKELV A0A0C7GD16 SEQ ID MDNRALLKGVRVVELSSFVAAPCCAKLLADWGAEVIKIEPLGGDGIRVMGGTFKSPCTDDENPMFEL NO: 56 ENGNKKGISVNVKTKEGVEILHKLLSKSDIFVTNVREKALAKMGLTYDQLKDDFPGLIHAHILGYGEE GPLKDKPGFDYTAYFARGGVSQSLMEKGTSPCNTAAGFGDHYAGISLTAGILAALYKKQITGEGDRV TVSLFHTALYGMGMMITTSQYGNEMPISRTEPNSPLMTTYKCKDGKWIQLALIQYNKWLPKFCEVIN RPEIMKDDRFNDIKVMPLHVDEMVKIVEKAMLEKTLDEWSDLLEEADLPFEKVQSCEDIINDDQAWA NDFLFKTTYENGNEGVLVNGPVKFKTMGIKEYEPAPRLGQHTEEVLKSIGYTEEEILDMVNSQAIKLD DAKELV A0A175L1W4 SEQ ID MTKEGLALEGVKVVELSSFVAAPSCSKLLADWGADVIKIEPIQGDNIRVVGGVYNSPARDDENPMFEL NO: 57 ENGNKRGIAINTRSEKGKEVLGKLLKDADVFVTNVREKALQRSGLSYDQLKDKYPSLIHAHILGYGEK GPLKDKPGFDYTAYFARGAVSTSLMEKGTSPANTNAGFGDHYAGMSLAAGILAALHRKTLTGKGDR VTVSLYHTAIFGMGLMITTAQYGNKMPLSRRTPNNPLATTYRCKDDRWIQLALLKYDAWFPKFCKEV INRPDLIEDLRFNKQSEVVKHVETFVGILEEEFIKKDLKEWADLLDKADLPYEKLQYCEDILEDEQAW ANDYLFKTTYDSGNTGVLVNSPVKFSEAGMRTYKAAPKIGEDTEVVLTSLGYSKEEIEEMRKEESIK A0A2X3BTQ9 SEQ ID MTKEGLALEGVKVVELSSFVAAPSCSKLLADWGADVIKIEPIQGDNIRVVGGVYNSPARDDENPMFEL NO: 58 ENGNKRGVAINTRSEKGKEVLGKLLKDADVFVTNVREKALQRSGLSYDQLKDKYPSLIHAHILGYGE KGPLKDKPGFDYTAYFARGAVSTSLMEKGTSPANTNAGFGDHYAGMSLAAGILAALHRKTLTGKGD RVTVSLYHTAIFGMGLMITTAQYGNKMPLSRRTPNNPLATTYRCKDDRWIQLALLKYDAWFPKFCKE VINRPDLIEDSRFNKQSEVVKHVETFVGVLEGEFIKKDLKEWADLLDKADLPYEKLQYCEDILEDEQA WANDYLFKTTYDSGNTGVLVNSPVKFSEAGMRPYKAAPKIGEDTEAILTSLGYSKEEIEEMRKENAIK Q5U924 SEQ ID MSEKKEARVVINDLLAEQYANAFKAKEEGRPVGWSTSVFPQELAEVFDLNVLYPENQAAGVAAKKG NO: 59 SLELCEIAESKGYSIDLCAYARTNFGLLENGGCEALDMPAPDFLLCCNNICNQVIKWYENISRELDIPLI MIDTTFNNEDEVTQSRIDYIKAQFEEAIKQLEIISGKKFDPKKFEEVMKISAENGRLWKYSMSLPADSSP SPMNGFDLFTYMAVIVCARGKKETTEAFKLLIEELEDNMKTGKSSFRGEEKYRIMMEGIPCWPYIGYK MKTLAKFGVNMTGSVYPHAWALQYEVNDLDGMAVAYSTMFNNVNLDRMTKYRVDSLVEGKCDG AFYHMNRSCKLMSLIQYEMQRRAAEETGLPYAGFDGDQADPRAFTNAQFETRIQGLVEVMEERKKL NRGEI A0A2X3BK09 SEQ ID MADKKEVKKNAAKMINGILAKSYADAWKAKEEGKPVGWSTSVFPQELVETFGLDVLYPENQAAGV NO: 60 AAKKESLSLCEAAESAGYSIDLCAYARTNFGLLEKGGSENLNMPKPDFICCCNNICNQVIKWYENIAK ELDIPLIMIDTTFNNEDEVTENRIKYLRAQFEEAIKQLEKISGKKFDPKKFEEVMKISAENGKLWKYSM SLPSGSFPSPMNGFDLFTYMAVIVCYRGKKETTEAFKLLISELEDNIKNKATSFRGEEKYRIMMEGIPC WPYIGYKMRTLAGYGVNMTGSVYPHAWALQYEVNDLDGMAKAYSTMFNNVNLETMCKYRIDSLID GNCDGAFYHMNRSCKLMSFIQYEMERKVFEETGIPYAGFDGDQADPRNFSKAQFETRLQGLVEVMEE RKKGGNK Q5U925 SEQ ID MYTMGLDIGSTASKGVILKNGEDIVASETISSGTGTTGPSRVLEKLYGKTGLAREDIKKVVVTGYGRM NO: 61 NYSDADKQISELSCHARGVNFIIPETRTIIDIGGQDAKVLKLDNNGRLLNFLMNDKCAAGTGRFLDVM AKIIEVDVSELGSISMNSQNEVSISSTCTVFAESEVISHLSENAKIEDIVAGIHTSVAKRVSSLVKRIGVQR NVVMVGGVARNSGIVRAMAREINTEIIVPDIPQLTGALGAALYAFDEAKESQKEVKNI A0A2X3BU19 SEQ ID MDNIKNILSKLEGIVKNPKKVVSDYKERTGNKVIGCFPVYTPEEIVYAADMLPIGIWGGDVEANLAKQ NO: 62 YYPAFCCSIMQSCMEFGLKGIYEGLSAVIIPGMCDTLNCMGQNWKFAIKDIPYIALVHPQNRKLEAGV EYLVEEYKHVKAKIEEIRGKEITEEEMQNSIDIYNEHRKVMRSFVDEAAKHPNTINNYQRNLVIKSGFF MRKDEHTKIVKELNELLAVLPEEKYDGKKVLVTGILLDSKEMLDVFEENKLRIVADDLAQESRQFRT DVPEGKNALDRLARQWSNIEGCSLAYDPKKIRGSMIAKEAKAKGIDGVVFAMMKFCDPEEYDYPIVK KDIEKEDIPTTMIEVDQQNKSVEQIRTRIQTFSEIL Q5U923 SEQ ID MEAILSKMKEVVENPNAAVKKYKSETGKKAIGCFPVYCPEEIIHAAGMLPVGIWGGQTELDLAKQYF NO: 63 PAFACSIMQSCLEYGLKGAYDELSGVIIPGMCDTLICLGQNWKSAVPHIKYISLVHPQNRKLEAGVKY LISEYKGVKRELEEICGYEIEEAKIHESIEVYNEHRKTMRDFVEVAYKHSNTIKPSIRSLVIKSGFFMRKE EHTELVKDLIAKLNAMPEEVCSGKKVLLTGILADSKDILDILEDNNISVVADDLAQETRQFRTDVPAG DDALERLARQWSNIEGCSLAYDPKKKRGSLIVDEVKKKDIDGVIFCMMKFCDPEEYDYPLVRKDIEDS GIPTLYVEIDQQTQNNEQARTRIQTFAEMMSLA A0A1V9IXA9 SEQ ID MYTMGLDIGSTTSKGVIIKDGEEIVASVLVPVGTGTSGPLKLIKELKEKSNLTEKDIEKTVVTGYGRIQY NO: 64 KDADKQISELSCHAKGVAFLIPGARTIIDIGGQDAKAMKLNDKGKLINFIMNDKCAAGTGRFLDVMA GVLETDVSKLGEISEKSTKEVSISSTCTVFAESEVISHLSANAKKEDIVAGIHTSVVRRVSTLAMRVGIE DQVVMVGGVARNKGIVKAMEKELGHDIKVPELAQLTGALGAAIYAFEETK Q73Q47 SEQ ID MIVKPMVRNNICLNAHPQGCKKGVEDQIEYTKKRITAEVKAGAKAPKNVLVLGCSNGYGLASRITAA NO: 65 FGYGAATIGVSFEKAGSETKYGTPGWYNNLAFDEAAKREGLYSVTIDGDAFSDEIKAQVIEEAKKKGI KFDLIVYSLASPVRTDPDTGIMHKSVLKPFGKTFTGKTVDPFTGELKEISAEPANDEEAAATVKVMGG EDWERWIKQLSKEGLLEEGCITLAYSYIGPEATQALYRKGTIGKAKEHLEATAHRLNKENPSIRAFVSV NKGLVTRASAVIPVIPLYLASLFKVMKEKGNHEGCIEQITRLYAERLYRKDGTIPVDEENRIRIDDWEL EEDVQKAVSALMEKVTGENAESLTDLAGYRHDFLASNGFDVEGINYEAEVERFDRI A0R484 SEQ ID MTSDVHDATDGVTETALDDEQSTRRIAELYATDPEFAAAAPLPAVVDAAHKPGLRLAEILQTLFTGY NO: 66 GDRPALGYRARELATDEGGRTVTRLLPRFDTLTYAQVWSRVQAVAAALRHNFAQPIYPGDAVATIGF ASPDYLTLDLVCAYLGLVSVPLQHNAPVSRLAPILAEVEPRILTVSAEYLDLAVESVRDVNSVSQLVVF DHHPEVDDHRDALARAREQLAGKGIAVTTLDAIADEGAGLPAEPIYTADHDQRLAMILYTSGSTGAP KGAMYTEAMVARLWTMSFITGDPTPVINVNFMPLNHLGGRIPISTAVQNGGTSYFVPESDMSTLFEDL ALVRPTELGLVPRVADMLYQHHLATVDRLVTQGADELTAEKQAGAELREQVLGGRVITGFVSTAPLA AEMRAFLDITLGAHIVDGYGLTETGAVTRDGVIVRPPVIDYKLIDVPELGYFSTDKPYPRGELLVRSQT LTPGYYKRPEVTASVFDRDGYYHTGDVMAETAPDHLVYVDRRNNVLKLAQGEFVAVANLEAVFSG AALVRQIFVYGNSERSFLLAVVVPTPEALEQYDPAALKAALADSLQRTARDAELQSYEVPADFIVETE PFSAANGLLSGVGKLLRPNLKDRYGQRLEQMYADIAATQANQLRELRRAAATQPVIDTLTQAAATIL GTGSEVASDAHFTDLGGDSLSALTLSNLLSDFFGFEVPVGTIVNPATNLAQLAQHIEAQRTAGDRRPSF TTVHGADATEIRASELTLDKFIDAETLRAAPGLPKVTTEPRTVLLSGANGWLGRFLTLQWLERLAPVG GTLITIVRGRDDAAARARLTQAYDTDPELSRRFAELADRHLRVVAGDIGDPNLGLTPEIWHRLAAEVD LVVHPAALVNHVLPYRQLFGPNVVGTAEVIKLALTERIKPVTYLSTVSVAMGIPDFEEDGDIRTVSPVR PLDGGYANGYGNSKWAGEVLLREAHDLCGLPVATFRSDMILAHPRYRGQVNVPDMFTRLLLSLLITG VAPRSFYIGDGERPRAHYPGLTVDFVAEAVTTLGAQQREGYVSYDVMNPHDDGISLDVFVDWLIRAG HPIDRVDDYDDWVRRFETALTALPEKRRAQTVLPLLHAFRAPQAPLRGAPEPTEVFHAAVRTAKVGP GDIPHLDEALIDKYIRDLREFGLI A0QWI7 SEQ ID MTIETREDRFNRRIDHLFETDPQFAAARPDEAISAAAADPELRLPAAVKQILAGYADRPALGKRAVEF NO: 67 VTDEEGRTTAKLLPRFDTITYRQLAGRIQAVTNAWHNHPVNAGDRVAILGFTSVDYTTIDIALLELGA VSVPLQTSAPVAQLQPIVAETEPKVIASSVDFLADAVALVESGPAPSRLVVFDYSHEVDDQREAFEAA KGKLAGTGVVVETITDALDRGRSLADAPLYVPDEADPLTLLIYTSGSTGTPKGAMYPESKTATMWQA GSKARWDETLGVMPSITLNFMPMSHVMGRGILCSTLASGGTAYFAARSDLSTFLEDLALVRPTQLNFV PRIWDMLFQEYQSRLDNRRAEGSEDRAEAAVLEEVRTQLLGGRFVSALTGSAPISAEMKSWVEDLLD MHLLEGYGSTEAGAVFIDGQIQRPPVIDYKLVDVPDLGYFATDRPYPRGELLVKSEQMFPGYYKRPEI TAEMFDEDGYYRTGDIVAELGPDHLEYLDRRNNVLKLSQGEFVTVSKLEAVFGDSPLVRQIYVYGNS ARSYLLAVVVPTEEALSRWDGDELKSRISDSLQDAARAAGLQSYEIPRDFLVETTPFTLENGLLTGIRK LARPKLKAHYGERLEQLYTDLAEGQANELRELRRNGADRPVVETVSRAAVALLGASVTDLRSDAHFT DLGGDSLSALSFSNLLHEIFDVDVPVGVIVSPATDLAGVAAYIEGELRGSKRPTYASVHGRDATEVRA RDLALGKFIDAKTLSAAPGLPRSGTEIRTVLLTGATGFLGRYLALEWLERMDLVDGKVICLVRARSDD EARARLDATFDTGDATLLEHYRALAADHLEVIAGDKGEADLGLDHDTWQRLADTVDLIVDPAALVN HVLPYSQMFGPNALGTAELIRIALTTTIKPYVYVSTIGVGQGISPEAFVEDADIREISATRRVDDSYANG YGNSKWAGEVLLREAHDWCGLPVSVFRCDMILADTTYSGQLNLPDMFTRLMLSLVATGIAPGSFYEL DADGNRQRAHYDGLPVEFIAEAISTIGSQVTDGFETFHVMNPYDDGIGLDEYVDWLIEAGYPVHRVD DYATWLSRFETALRALPERQRQASLLPLLHNYQQPSPPVCGAMAPTDRFRAAVQDAKIGPDKDIPHVT ADVIVKYISNLQMLGLL D6Z860 SEQ ID MTQSHTQGPQASAAHSRLARRAAELLATDPQAAATLPDPEVVRQATRPGLRLAERVDAILSGYADRP NO: 68 ALGQRSFQTVKDPITGRSSVELLPTFDTITYRELRERATAIASDLAHHPQAPAKPGDFLASIGFISVDYV AIDIAGVFAGLTAVPLQTGATLATLTAITAETAPTLFAASIEHLPTAVDAVLATPSVRRLLVFDYRAGS DEDREAVEAAKRKIADAGSSVLVDVLDEVIARGKSAPKAPLPPATDAGDDSLSLLIYTSGSTGTPKGA MYPERNVAHFWGGVWAAAFDEDAAPPVPAINITFLPLSHVASRLSLMPTLARGGLMHFVAKSDLSTL FEDLKLARPTNLFLVPRVVEMLYQHYQSELDRRGVQDGTREAEAVKDDLRTGLLGGRILTAGFGSAP LSAELAGFIESLLQIHLVDGYGSTEAGPVWRDGYLVKPPVTDYKLIDVPELGYFSTDSPHPRGELAIKT QTILPGYYKRPETTAEVFDEDGFYLTGDVVAQIGPEQFAYVDRRKNVLKLSQGEFVTLAKLEAAYSSS PLVRQLFVYGSSERSYLLAVIVPTPDALKKFGVGEAAKAALGESLQKIARDEGLQSYEVPRDFIIETDPF TVENGLLSDARKSLRPKLKEHYGERLEAMYKELADGQANELRDIRRGVQQRPTLETVRRAAAAMLG ASAAEIKPDAHFTDLGGDSLSALTFSNFLHDLFEVDVPVGVIVSAANTLGSVAEHIDAQLAGGRARPTF ATVHGKGSTTIKASDLTLDKFIDEQTLEAAKHLPKPADPPRTVLLTGANGWLGRFLALEWLERLAPAG GKLITIVRGKDAAQAKARLDAAYESGDPKLAGHYQDLAATTLEVLAGDFSEPRLGLDEATWNRLAD EVDFISHPGALVNHVLPYNQLFGPNVAGVAEIIKLAITTRIKPVTYLSTVAVAAGVEPSALDEDGDIRT VSAERSVDEGYANGYGNSKWGGEVLLREAHDRTGLPVRVFRSDMILAHQKYTGQVNATDQFTRLVQ SLLATGLAPKSFYELDAQGNRQRAHYDGIPVDFTAESITTLGGDGLEGYRSYNVFNPHRDGVGLDEFV DWLIEAGHPITRIDDYDQWLSRFETSLRGLPESKRQASVLPLLHAFARPGPAVDGSPFRNTVFRTDVQK AKIGAEHDIPHLGKALVLKYADDIKQLGLL P39135 SEQ ID MKIYGIYMDRPLSQEENERFMSFISPEKREKCRRFYHKEDAHRTLLGDVLVRSVISRQYQLDKSDIRFS NO: 69 TQEYGKPCIPDLPDAHFNISHSGRWVICAFDSQPIGIDIEKTKPISLEIAKRFFSKTEYSDLLAKDKDEQT DYFYHLWSMKESFIKQEGKGLSLPLDSFSVRLHQDGQVSIELPDSHSPCYIKTYEVDPGYKMAVCAAH PDFPEDITMVSYEELL AB213459.1 SEQ ID MAQYDVADRSAIVTGGGSGIGRAVALTLAASGAAVLVTDLNEEHAQAVVAEIEAAGGKAAALAGDV NO: 70 TDPAFGEASVAGANALAPLKIAVNNAGIGGEAATVGDYSLDSWRTVIEVNLNAVFYGMQPQLKAMA ANGGGAIVNMASILGSVGFANSSAYVTAKHALLGLTQNAALEYAADKVRVVAVGPGFIRTPLVEANL SADALAFLEGKHALGRLGEPEEVASLVAFLASDAASFITGSYHLVDGGYTAQ Q84H78 SEQ ID MRVFAVQPEDTTIHDLQVPTPSPEGREVLLRVVRAGVCHTDTHLRAGGYDLGSRGMMSMKERGIEYP NO: 71 MVLGHEVVGVVEKVGDGVESVQVGDIRLIYPWIGCGECRQCRAGHDNRCAAGKNLGVARHGGYAE NILVPDEKYLVDIDGLDPSWAATLACSGLTAYSAVDKALPLEPDEPVVVFGAGGLGLTAIAILRSRGH RNICAVDVAERNLALARDMGASSTVLSGTGSGADDIRGAAGGPAGAVIDFVNNGATATTAFEVLAKA GIMIQVGLFGGEVTLPTALLALRMIRIEGSFVGTLVQMQDLVRLAQRGELPHIPVVERSLSAAAVSQAL DDLTAGGVAGRIVLTA Q7WVD0 SEQ ID MHCYCVTHHGQPLEDVEKEIPQPKGTEVLLHVKAAGLCHTDLHLWEGYYDLGGGKRLSLADRGLKP NO: 72 PLTLSHEITGQVVAVGPDAESVKVGMVSLVHPWIGCGECNYCKRGEENLCAKPQQLGIAKPGGFAEYI IVPHPRYLVDIAGLDLAEAAPLACAGVTTYSALKKFGDLIQSEPVVIIGAGGLGLMALELLKAMQAKG AIVVDIDDSKLEAARAAGALSVINSRSEDAAQQLIQATDGGARLILDLVGSNPTLSLALASAARGGHIV ICGLMGGEIKLSIPVIPMRPLTIQGSYVGTVEELRELVELVKETHMSAIPVKKLPISQINSAFGDLKDGN VIGRIVLMHEN D8GL45 SEQ ID MENFIFKNATEIIFGKDTENLVGSKVKEYSKSDKILFCYGGGSIKRSGLYDRVIKSLKENGIEFIELPGIK NO: 73 PNPRLGPVKEGIRLCRENNIKFVLSVGGGSSADTAKAIAVGVPYKGDVWDFYTGKAEVKEALPVGVV ITLPATGTESSNSSVIMNEDGWFKKGLNTVLIRPAFSIMNPELTFTLPEYQTACGACDIMAHIMERYFTN VKHVDITDRLCEAALRNVINNAPIVLKDPKNYDARAEIMWTGTIAHNDVLSAGRIGDWASHKIEHELS GETDIAHGAGLAIVFPAWMKYVYKHDINRFVQFAVRVWDVDLSYSSCEDIVLEGIRRMTAFFKSMGL PVTLKEGSIGEDKIEEMANKCTDNGTKTVGQFVKLNKDDIVKILNLAK Q04944 SEQ ID MLSFDYSIPTKVFFGKGKIDVIGEEIKKYGSRVLIVYGGGSIKRNGIYDRATAILKENNIAFYELSGVEPN NO: 74 PRITTVKKGIEICRENNVDLVLAIGGGSAIDCSKVIAAGVYYDGDTWDMVKDPSKITKVLPIASILTLSA TGSEMDQIAVISNMETNEKLGVGHDDMRPKFSVLDPTYTFTVPKNQTAAGTADIMSHTFESYFSGVEG AYVQDGIAEAILRTCIKYGKIAMEKTDDYEARANLMWASSLAINGLLSLGKDRKWSCHPMEHELSAY YDITHGVGLAILTPNWMEYILNDDTLHKFVSYGINVWGIDKNKDNYEIAREAIKNTREYFNSLGIPSKL REVGIGKDKLELMAKQAVRNSGGTIGSLRPINAEDVLEIFKKSY Q9R2F4 SEQ ID MNYPNIPLYINGEFLDHTNRDVKEVFNPVNHECIGLMACASQADLDYALESSQQAFLRWKKTSPITRS NO: 75 EILRTFAKLAREKAAEIGRNITLDQGKPLKEAIAEVTVCAEHAEWHAEECRRIYGRVIPPRNPNVQQLV VREPLGVCLAFSPWNFPFNQAIRKISAAIAAGCTIIVKGSGDTPSAVYAIAQLFHEAGLPNGVLNVIWG DSNFISDYMIKSPIIQKISFTGSTPVGKKLASQASLYMKPCTMELGGHAPVIVCDDADIDAAVEHLVGY KFRNAGQVCVSPTRFYVQEGIYKEFSEKVVLRAKQIKVGCGLDASSDMGPLAQARRMHAMQQIVED AVHKGSKLLLGGNKISDKGNFFEPTVLGDLCNDTQFMNDEPFGPIIGLIPFDTIDHVLEEANRLPFGLAS YAFTTSSKNAHQISYGLEAGMVSINHMGLALAETPFGGIKDSGFGSEGGIETFDGYLRTKFITQLN Q8GEZ8 SEQ ID MISKGFSTQTERINILKAQILNAKPCVESERAILITESFKQTEGQPAILRRALALKHILENIPITIRDQELIV NO: 76 GSLTKEPRSSQVFPEFSNKWLQDELDRLNKRTGDAFQISEESKEKLKDVFEYWNGKTTSELATSYMTE ETREAVNCDVFTVGNYYYNGVGHVSVDYGKVLRVGFNGIINEAKEQLEKNRSIDPDFIKKEKFLNSVII SCEAAITYVNRYAKKAKEIADNTSDAKRKAELNEIAKICSKVSGEGAKSFYEACQLFWFIHAIINIESNG HSISPARFDQYMYPYYENDKNITDKFAQELIDCIWIKLNDINKVRDEISTKHFGGYPMYQNLIVGGQNS EGKDATNKVSYMALEAAVHVKLPQPSLSVRIWNKTPDEFLLRAAELTREGLGLPAYYNDEVIIPALVS RGLTLEDARDYGIIGCVEPQKPGKTEGWHDSAFFNLARIVELTINSGFDKNKQIGPKTQNFEEMKSFDE FMKAYKAQMEYFVKHMCCADNCIDIAHAERAPLPFLSSMVDNCIGKGKSLQDGGAEYNFSGPQGVG VANIGDSLVAVKKIVFDENKITPSELKKTLNNDFKNSEEIQALLKNAPKFGNDIDEVDNLAREGALVY CREVNKYTNPRGGNFQPGLYPSSINVYFGSLTGATPDGRKSGQPLADGVSPSRGCDVSGPTAACNSVS KLDHFIASNGTLFNQKFHPSALKGDNGLMNLSSLIRSYFDQKGFHVQFNVIDKKILLAAQKNPEKYQD LIVRVAGYSAQFISLDKSIQNDIIARTEHVM Q8GEZ7 SEQ ID MSKEIKGVLFNIQKFSLHDGPGIRTIVFFKGCSMSCLWCSNPESQDIKPQVMFNKNLCTKCGRCKSQCK NO: 77 SAAIDMNSEYRIDKSKCTECTKCVDNCLSGALVIEGRNYSVEDVIKELKKDSVQYRRSNGGITLSGGE VLLQPDFAVELLKECKSYGWHTAIETAMYVNSESVKKVIPYIDLAMIDIKSMNDEIHRKFTGVSNEIIL QNIKLSDELAKEIIIRIPVIEGFNADLQSIGAIAQFSKSLTNLKRIDLLPYHNYGENKYQAIGREYSLKELK SPSKDKMERLKALVEIMGIPCTIGAE A5VMB2 SEQ ID MKRQKRFEELEKRPIHQDTFVKEWPEEGFVAMMGPNDPKPSVKVENGKIVEMDGKKLEDFDLIDLYI NO: 78 AKYGINIDNVEKVMNMDSTKIARMLVDPNVSRDEIIEITSALTPAKAEEIISKLDFGEMIMAVKKMRPR RKPDNQCHVTNTVDNPVQIAADAADAALRGFPEQETTTAVARYAPFNAISILIGAQTGRPGVLTQCSV EEATELQLGMRGFTAYAETISVYGTDRVFTDGDDTPWSKGFLASCYASRGLKMRFTSGAGSEVLMGY PEGKSMLYLEARCILLTKASGVQGLQNGAVSCIEIPGAVPNGIREVLGENLLCMMCDIECASGCDQAY SHSDMRRTERFIGQFIAGTDYINSGYSSTPNYDNTFAGSNTDAMDYDDMYVMERDLGQYYGIHPVKE ETIIKARNKAAKALQAVFEDLGLPKITDEEVEAATYANTHDDMPKRDMVADMKAAQDMMDRGITAI DIIKALYNHGFKDVAEAILNLQKQKVVGDYLQTSSIFDKDWNVTSAVNDGNDYQGPGTGYRLYEDK EEWDRIKDLPFALDPEHLEL A5VMB1 SEQ ID MADIDENLLRKIVKEVLSETNQIDTKIDFDKSNDSTATATQEVQQPNSKAVPEKKLDWFQPVGEAKPG NO: 79 YSKDEVVIAVGPAFATVLDKTETGIPHKEVLRQVIAGIEEEGLKARVVKVYRSSDVAFCAVQGDHLSG SGIAIGIQSKGTTVIHQKDQDPLGNLELFPQAPVLTPETYRAIGKNAAMYAKGESPEPVPAKNDQLARI HYQAISAIMHIRETHQVVVGKPEEEIKVTFD A5VMB0 SEQ ID MMSEVDDLVAKIMAQMGNSSSANSSTGTSTASTSKEMTADDYPLYQKHRDLVKTPKGHNLDDINLQ NO: 80 KVVNNQVDPKELRITPEALKLQGEIAANAGRPAIQKNLQRAAELTRVPDERVLEMYDALRPFRSTKQE LLNIAKELRDKYDANVCAAWFEEAADYYESRKKLKGDN A5VMA9 SEQ ID MATEKVIGVDIGNSSTEVALADVSDSGQVHFINSGIAPTTGIKGTKQNLVGIRDSITQVLNKSNLTIDDI NO: 81 DLIRINEATPVIGDVAMETITETVVTESTMIGHNPNTPGGIGTGAGITVRLLDLLKKTDKSKNYIVVVPK DIDFEDVAKLINAYVASGYKITAAILRNDDGVLVDNRLNHKIPIVDEVAMIDKVPLNMLAAVEVAGPG QVISQLSNPYGIATLFGLTPEETKNIVPVSRALIGNRSAVVIKTPAGDVKARVIPAGKIIINGDTGKEEVG VSEGADAIMKKVSSFRHINNITGESGTNVGGMLENVRQTMADLTGKKNDEIAIQDLLAVDTQVPVEV RGGLAGEFSNESAVGIAAMVKSDHLQMEVIAKLIEKEFNTKVEIGGAEVESAIRGALTTPGTDKPIAIL DLGAGSTDASIINKENNTVAIHLAGAGDMVTMIINSELGLNDIHLAEDIKRYPLAKVENLFQIRHEDGS VQFFKDPLPSSLFAKVVVIKPDGYEPVTGNPSIEKIKLVRQSAKKRVFVTNALRALKYVSPTGNIRDIPF VVIVGGSALDFEIPQLVTDELAHFNLVAGRGNVRGVEGPRNAVATGLILRYGEERRKRYEQR A5VMA8 SEQ ID MNNDDSQRPSIVVGLENGITIPDSVKPLFYGIEEEQIPVSVRKININDTVERAYQSALASRLSVGIAFEGD NO: 82 HFIVHYKNLKENQPLFDMTINDKKQLRILGANAARLVKGIPFKEMANR Q6QBS4 SEQ ID MYTVGDYLLDRLHELGIEEIFGVPGDYNLQFLDQIISREDMKWIGNANELNASYMADGYARTKKAAA NO: 83 FLTTFGVGELSAINGLAGSYAENLPVVEIVGSPTSKVQNDGKFVHHTLADGDFKHFMKMHEPVTAAR TLLTAENATYEIDRVLSQLLKERKPVYINLPVDVAAAKAEKPALSLEKESSTTNTTEQVILSKIEESLKN AQKPVVIAGHEVISFGLEKTVTQFVSETKLPITTLNFGKSAVDESLPSFLGIYNGKLSEISLKNFVESADF ILMLGVKLTDSSTGAFTHHLDENKMISLNIDEGIIFNKVVEDFDFRAVVSSLSELKGIEYEGQYIDKQYE EFIPSSAPLSQDRLWQAVESLTQSNETIVAEQGTSFFGASTIFLKSNSRFIGQPLWGSIGYTFPAALGSQI ADKESRHLLFIGDGSLQLTVQELGLSIREKLNPICFIINNDGYTVEREIHGPTQSYNDIPMWNYSKLPETF GATEDRVVSKIVRTENEFVSVMKEAQADVNRMYWIELVLEKEDAPKLLKKMGKLFAEQNK KMK64081.1 SEQ ID NO: MSAKRTLLTVDDVTGCWAIMPTPAKDDASDWRTEFSVDLDETARVANALVESGVDGILALGTFGEG 84 ATLTWEEKEAYVRTVVDAVAGRVPFFAGTTSLNTRETIRQMRIVRDIGVDGVMLGIPMWVEADTATA VQFYRDVTEACPDVAICAYANPEAFKYEFGRAFWAQVSDLPQIVSAKYLNMGGLYPDLNLSKRRIRL MPLDVDYYAAARIDPDHCTAFWTSGAVCGPEPAILLRDLMEKARKSGDWAEAKALTDRIGMTYKTL FPNGSFKEFSRYNISIEKIRMDAAGWMKAGPCRPPYHVTPEPILEGGRIAGQKWAELAESLRAGN WP_ SEQ ID NO: MITAAEINGMYGIIPTPALPGAERLDARDTVDVDETARVVDRLIRDGVSGIIALGTTGECPALSEDDFD 070028041.1 85 VVTDTVVEAVAGRVPVFVGATGAGGHGTARRLRKVAASGATGALLGLPMWQPLTTAMAVEYYAQ ASAAFPDLALMVYANARAFRYTFPVEFWQGVSSQAPTVTSAKVSRAPQLERMLEVTGKKVNFIPSDM VVHDFAARAPQTTTACWATAAGMGPEPSIALMDALRRGDSEAAGRAVAGIAWANEPLAHLFADQEI FASYNTQIEKSRIAAAGYCRPGPVRSPYHHLPEEYAAASAVCGQRWRELRERIAAGTNDQK KZL92449.1 SEQ ID NO: MIKGYSLPLTPKGTSNIVPAPPWHYVGNVLAIEYEAYAENIAAFLPEGLEFSSNQCAIYFIEWQYCSEFG 86 EEHLDPVNSQYKETIVLVSANYKGTPVSYCPFIWVDQDLSLMRGLIQGWPKQLGETYITRPYNLPSKA ASNLEKGGKLGATLSVKGRRLVDARITVNKKTETLPNPTFAQAINLRHFPELVLGRHNQPLIHELVQL KSRDLHISPIWKGDAILNFFDHPFIELSDLKPTKVKNSYYFSAALTVDDLSQLEV A0A1G9R408 SEQ ID NO: MRAVVVRSHGGPEVLVAEELDRPEPGPGAVLVDVAAAGVNYIDTYHREGVYPIPTPFTLGLEGAGTV 87 AALGEGVTEFAVGDRVAWASAIGSYAQQVAAPAAQLVPVPSTVDLEIAAGAMLQGMTAHYLTASTH PIAEGDVALVHAAAGGMGLLLTQMIKARGGRVIGTVSTAEKEKLAREAGADEVIRYTEQDVAQRVRE LTDGVGVHVVYDGVGKDTFDASLASLRPRGLLALYGAASGAVPPFDAQRLNAGGSLFLTRPSLGHHT ATREELLWRAGEVFDAIQAGELDIAIGGRYALDSARQAHEDLQGRRTTGKLLLTTS G4Q8R5 SEQ ID NO: MKAIVMKEFGGPEVLKYVDVPDPVPEANEVLIKLAFCGVNPNETYVRTGTYNFYKPELPYTPGYDGA 88 GVIEKVGAGVTHVKVGDRVFVAALLAKRNTGTYAQKVVCDADSVHKLPDFISFEEGASFGIPAMAAY RALFHRAHIKAGEIVMIHGAEGGVGSLAVQMAKAVGAIVIGTGTTPEGLDIVRSFGADYAIYHLKADN QDELMELTKGKGPDVIIEFLANVNLQTDLKVIAKYGRIVVVGNRGTIEINPRLAMANESTILGMALWN APANEYRESLFALRAFMQSGAVRAKVGKQLLLKDAAQAHNEIINGLAKGKMILKIE ANA98723.1 SEQ ID NO: MRAIEVPVTGGPEVLTLVEKTAPTPGPGEVLIDVDAVGVNFRDIYLRNGSYAAPLPHIPGSEVTGVVSA 89 VGEGVENLAPGDRVASPVAAWGYAESTTAPADYTAKVPAGLSSEVAASALLQGITAHYLLTSVYPVA AGDTVLVHAGAGGMGLLLTQWASHRGVRVITTVSSAAKEKLSREAGAAEVLPYPDPTDPAEFAEKIL ELTSGEGVAVAYDGVGKSTFEASLAAVRVRGLIALYGAASGQVPPFDPQRLTAKSAVLTRPTMGHFIR TPAEFAWRADDVLDLVSRGTLKITVGASYPLEQAAQAHIDLEARKTTGSVVLVP K0EUQ3 SEQ ID NO: MRAIQVSEHGGPEVLHHVELPDPTIDADQLLVDVQATGINFIDTYIRTGRYPQDVPYVPGSEATGVVA 90 EVGANVTEFAVGDRVAWASAPGSYAERVAVRADVAVEVPDGVEPPVAASALLQGMTAHYLLESIYT PEPGETVLVHAGAGGVGLILTQLAVARGARVITTVSSDVKEKLSREAGATEVLRYGDDLADEVRTLT DGVGVAAVYDGVGASTFEASLRSLRVRGMLALFGAASGPVPPFDLQRLNGAGSLFVTRPSLAFYTRD RAELLWRATDIFTAIAEGTLQIRIGATYPLAEAEQAHRDLESRKTTGSIVLLP A0A061CRS8 SEQ ID NO: MAKRIQFSQHGGSEVLEYRDYQPAAPGPREVRVANKAIGLNFIDTYFRSGLYQPPALPSSLGTEGAGV 91 VEAIGSEVEGLKVGDRVAYATGPLGAYSELHVLPADNLVHLPDSISFEQAAAVMLKGLTVQYLLRQT YELKGGETILFHAAAGGVGSFACQWAKALGVNLIGTVSSAKKAALAKELGAWETIDYSHENVVQRV LELTDGAKCPVVYDGVGKDTWETSLDCVAPRGLLVSFGNASGAVTGVNLGILAQKGSLYVTRPTLAS YANTPQNLQAMADELFAMISSGKLQVDISNRYALKDAAAAQDALSSRQTTGSTILLP Q9A212 SEQ ID NO: MLAVQAVRTGGPEVLEVVDLPLPSPGPGQILVRHQAVGLNYIDTYHRSGLYPVKTPLVIGLEAAGVVE 92 SVGEAVTRFKVGDRVAYNGTMGAYAQAAVVPAERAVLVPDGVSLEVAAAALLKGMTAEFLVRRCF HVKQGDWVLVHAAAGGVGQILVQWCKALGATVVATVGSTAKATIARDLGADHVIDYSHEDVAARV AELTGGRGVAVVYDGVGKDTWEASLASLARRGMLVTFGNASGPAPAFPPLALAPKSAFVTRPKLFD YIVTTEELDESAQALFAVIASGAIKIDIGQTFPLAEARAAHEALEGRRTTGATLLLP A0A1I6RWW2 SEQ ID NO: MRAIRVTSHGGPEALEVSEVEVPEPGPGQLLVDVAASGVNFIDTYQRSGVYSVPLPFTPGSEGAGEIVA 93 VGPDVDGFAVGERVAWAMTPGSYAEKALVPARAAVKIPDGVDTRTAAAATLQGMTAHFLVTSTHEI KTGETALVHAAAGGMGLLLTQLIKSKGGNVIGTVSTDEKERLAREAGADEIIRYTEADVAAEVKDLT DGRGVDVVYDGVGKSTFEASLASLRPRGTLALFGGASGQVPPFDPQRLNGAGSLFLTRPSLAHHVLTR EELEWRAGEVFGWISSGALHIRVSGTYSLEDAARAHEDLEGRRTTGKLLILP WP_ SEQ ID NO: MTNAIRVHETGGPEVLRLDEVTREAGAGQLLVRVEAAGVNFIDTYQRSGVYSVELPHALGLEGAGTV 026197277.1 94 EAVGDEASDFTPGDRVAWVWAAGSYAEHTVVPVERAVRIPDDVDTKTAGALMLQGLTAHYLLRST YRVDETDTVLVHAAAGGVGLLLVQLAKSLGARVIATASTAEKRALATGAGADEVLGYEGFDTKLRE LTGGIGVSVVYDGVGKDTFDASLASIRPRGYLVLFGGSSGQVPPFDLQRLNAAGSLFVTRPSLGPYIAD RTEYEWRVGELFEAVGNGSLNVRIGGSYPLAEAANAHRDLEGRKTTGKLLLVP Q5NKZ3 SEQ ID NO: MSEAYAIIAEKAGGPEVLVKKPLDLGKMKPEAGQVLLRHQAIGLNFIDIYHRSGLYKQDFPANLGCEA 95 AGVIEWGDKVKGFKAGDRVAVFTSKPGAYATHRIVDASELVALPDDISAETAAAVLLKGMTSWML AEKCLAHAAIEGEAPKVMVLAAAGGVGSLLIPWLKYLGVTVFAHTSTEEKAAKVKANGADYVTTLP YSDLPDWVRKQNHGEGVHAVLDSVGADSWKSSIASLRKKGLWVVYGNASGPVPALSPLELSKAGSI YTSRPRLIDYVDNSVDLTTASQKLFALLRKNILKVEINQRFPLTEVAKAHQLLESRKTTGSTVLIP WP_ SEQ ID NO: MPKAIRVHEYGGPEVMRYEEVDLPAPGPGQIRVRQRAVGVNFIDIYFRSGLYKAPQLPFTPGNEGTGE 012333034.1 96 VVAVGEGVAGLAVGDRVAYGSAAQTYAQEAVIEARMAVKVPDGIDDATAAAMMLKGLTAQYLLR KTYRVQPGDTILFHAAAGGVGLIATQWAKHLGATVIGTVGSRDKAELAKQHGCDHVILYRDEDFAA RVKEITGGKGCAVVYDGVGQATYPASLDCLRPFGMFVSFGNASGVIENFNIGLLGPKGSLYATRPTLF THVAERASLEAMADDLFGVVGSGAVRIPVHSRVPLAEAAQVHRDLAGRQTTGATVLIP WP_ SEQ ID NO: MAKAIRFEKTGGPEVMQWVDVEVGDPGSGEVRIKQHAVGLNYIDVYFRTGLYPMPLPGGLGMEAAG 136898000.1 97 EVTAVGPDVEGLRVGDRVAYVARPPGAYAQERVLPAAALVKLPGALGYDDAASAMLQGLTAQYLL RRTYRVKAGDTILIQAAAGGVGLFVCQWAKALGATVIGTVSSDEKAELAKAHGCDYPIVYTRESFTK RVKEITGGAGVPVVYDSIGKDTFTGSLDCLAPLGLFVSFGNASGPLPPIDSSEFAGRGSLFFTRPTLFTHI AKRSDYDAMAAELFDVIVSGKVKTMIRQRFPLAEVGQAHADLEARRTTGSTILIP WP_ SEQ ID NO: MKILVFGARDYEEPVIKKWSEEHKDVQVDIYPENMTEENVVKAKGYDGISIQQTNYIDNPYIYETLKD 003431407.1 98 AGVKVIASRTAGVDMIHFDLVNENGLIVTNVPSYSPNAIAELAVTQAMNLLRKTPLVKKKVCEGDYR WIAELLGTEVRSITVGVIGTGKIGATSAKLFKGLGANVIAFDQYPNSDLNDILTYKDSLEDLLKEADLIT LHTPLLEGTKHMINKDTLAIMKDGAYIVNTGRGGLINTGDLIEALESGKIRAAALDTFETEGLFLNKKM NPGELTDPEINKLLSMEQVIFTHHLGFFTSTAIENIVYSSLSSAVEVIKTGTATNRVN BAL51292.1 SEQ ID NO: MRITIAGAGAMGSRFGLMLHKGGNEVTLIDGWPEHVKAIKDHGLRANYNGEELTAHLSVELQSEISS 99 KEKTDLIILFTKAMQLDKMLQDIKPLIDEHTKVLCLLNGIGHEDTIEKYVSKNNIFIGNTMWTAGLEGP GKAKLFGDGSVELQNLISGEEETAKKLAEILSESGLNAKYSNNIHYSIYRKACVNGTMNGLCTILDTN MAGLGETKPAHDMVVTIVNEFAAVAKFENVNLDIAEVVQHVETCFDPATIGLHYPSMYQDLIKNNRL TEIDYINGAVSRKGKKYNVATPYCDFLTQLVHSKEELLKAK AKC64094.1 SEQ ID NO: MKILMYSVREHEKPAIKKWLEANPGVQIDLSDEALSEDTVCKVKDYDGIAIQQTNSIGGETVYSTLKK 100 YGIRQIASRTAGVDMIDLKMASENNIIVTNVPAYSPNAIAELAVTHTMNLLRNIKTVNKRIAFGDYRW SADLIAREVRSITVGVVGTGKIGRTSAKLFKGLGANVIGYDAYPDKKLEENNLLTYKDSLEDLLKEAD VVTLHTPLLESTKHMINKNNLKYMKPNAFIVNTGRGGIINTEDLIEALEENKIAGAALDTFENEGLFLN KVIDPTKIPDPQLDKLLKMDQVLITHHVGFFTTTAVQNMVDTSLDSVMEVLKTNDSVNKAN WP_ SEQ ID NO: MTKIAMYNVSPIEVPYIEDWAKKNDVEIKTTDQALTSATVDLAEGCSSVSLKPLGPVDEEVVYQKLSE 002876862.1 101 YGVKCIGLRIVGFNTINFDWTKKYNLLVTNVPVYSPRAIAEMTVTQAMYLLRKIGEFRYRMDHDHDF TWPSNLISNEIYNLTVGLIGVGHIGSAVAEIFSAMGAKVIAYDVAYNPEFEPFLTYTDFDTVLKEADIVS LHTPLLPSTENMIGEKQLKEMKKSAYLINCARGELVDTGALIKALQDGEIAGAGLDTLAGESSYFGHT GLTDSEIPEDYKTLAKMPNVVITPHSAFYTETSIRNMVQICLTDQLTIAKGGRPRSIVNL AGP69017.1 SEQ ID NO: MTKILMYTVRPDERAAIDAWVAANDIQVDTNTVEFGPDTVDLAKGYDGVVIQQHGAIPEEMVYQKL 102 KAFGIKQLTLRITGYDIVNLDAATANGLVVTNVPAYSPRSVSELVLAQVMRLIRHLGEASAREAKDDY SWTGLEAPEIHNLTVGIIGAGKIGSAVARIFRALGATVIVSDPVKRPELADTVSYVDLNTLLTTSDVVT VHTPLDGLTTHLIDADALRKMKSTAYLINAARGPIVDTEALIKALNDHTIAGAALDTIEGEAGIFGEDR SQTLVDNQTLETLKAMPNVEISPHIGFYTDAAVKNMIDISLDDVKTILEGGKSAHQVN WP_ SEQ ID NO: MKIIAYAVRDDERPFFDTWMKENPDVEVKLVPELLTEDNVDLAKGFDGADVYQQKDYTAEVLNKLA 003640741.1 103 DEGVKNISLRNVGVDNLDVPTVKARGLNISNVPAYSPNAIAELSVTQLMQLLRQTPLFNKKLAKQDFR WAPDIAKELNTMTVGVIGTGRIGRAAIDIFKGFGAKVIGYDVYRNAELEKEGMYVDTLDELYAQADV ITLHVPALKDNYHMLNADAFSKMKDGAYILNFARGTLIDSEDLIKALDSGKVAGAALDTYEYETKIFN KDLEGQTIDDKVFMNLFNRDNVLITPHTAFYTETAVHNMVHVSMNSNKQFIETGKADTQVKFD AKC64095.1 SEQ ID NO: MKILAYCVRPDEIDSFKNFSEKYGHTVDLIPDSFGPSVAHLAKGYDGISILGNDTCNREALEKIKDCGIK 104 YLATRTAGVNNIDFDAAKEFGINVANVPAYSPNSVSEFTVGLALSLTRKIPFALKRVELNNFALGGLIG VELRNLTLGVIGTGRIGLKVIEGFSGFGMKKMIGYDIFENEKAKEYIEYKSLDEVYKEADIITLHAPLTD DNYHMIGKESIAKMKDGVFIINAARGALIDSEALIEGLKSGKIAGAALDSYEYEQGVFHNNKMNEIMK DDTLARLKSFPNVVITPHLGFYTDEAVSNMVEITLMNLQEFELKGTCKNQRVCK AKC64094.1 SEQ ID NO: MKILMYSVREHEKPAIKKWLEANPGVQIDLSDEALSEDTVCKVKDYDGIAIQQTNSIGGETVYSTLKK 105 YGIRQIASRTAGVDMIDLKMASENNIIVTNVPAYSPNAIAELAVTHTMNLLRNIKTVNKRIAFGDYRW SADLIAREVRSITVGVVGTGKIGRTSAKLFKGLGANVIGYDAYPDKKLEENNLLTYKDSLEDLLKEAD VVTLHTPLLESTKHMINKNNLKYMKPNAFIVNTGRGGIINTEDLIEALEENKIAGAALDTFENEGLFLN KVIDPTKIPDPQLDKLLKMDQVLITHHVGFFTTTAVQNMVDTSLDSVMEVLKTNDSVNKAN 

1. A method comprising: contacting pyruvate and an aliphatic aldehyde with an aldol-dehydration product biosynthesis polypeptide so that an aldol-dehydration product is produced, wherein: the carbonyl group of the aliphatic aldehyde is not conjugated to a alkenyl, alkynyl, or aromatic group; and the aldol-dehydration product is a compound comprising an aldehyde or ketone group and a double bond conjugated with the aldehyde or ketone group.
 2. The method of claim 1, wherein the aldol-dehydration product biosynthesis polypeptide is or comprises a hydratase-aldolase.
 3. The method of claim 1, wherein the aldol-dehydration product biosynthesis polypeptide is or comprises an enzyme having an EC number 4.1.2.45 or EC number 4.1.2.34, or EC 4.1.1.4, or is selected from Tables 1 and 5-8.
 4. (canceled)
 5. A method comprising: contacting an alkene with an alkene reduction product biosynthesis polypeptide so that an alkene reduction product is produced, wherein: the alkene reduction product biosynthesis polypeptide is or comprises an enzyme that belongs to EC 1.6.5 or is selected from Table 9; the alkene comprises a double bond conjugated to a carbonyl group; and a double bond conjugated to a carbonyl group in the alkene is reduced to a single bond to provide an alkene reduction product. 6-7. (canceled)
 8. The method of claim 2, wherein the aliphatic aldehyde has the structure of formula A-1 thereof: R^(a)-L²-L¹-C(O)H,   A-1 or a salt thereof, wherein: R^(a) is R″ or —OR″, each of L¹ and L² is independently a covalent bond, or a bivalent, optionally substituted, linear or branched C₁₋₂₀ aliphatic or C₁₋₂₀ heteroaliphatic, wherein one or more methylene units are optionally and independently replaced by —C≡C—, —C(R″)₂—, -Cy-, —O—, —S—, —S—S—, —N(R″)—, —C(O)—, —C(S)—, —C(NR″)—, —C(O)N(R″)—, —N(R″)C(O)N(R″)—, —N(R″)C(O)O—, —S(O)—, —S(O)₂—, —S(O)₂N(R″)—, —C(O)S—, or —C(O)O—; -Cy- is a bivalent, optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring, wherein each monocyclic ring is independently an optionally substituted, saturated, partially saturated or aromatic 3-20 membered ring having 0-5 heteroatoms; each R″ is independently —R′, —C(O)R′, —CO₂R′, or —SO₂R′; R′ is hydrogen, or an optionally substituted group selected from C₁₋₁₀ aliphatic, C₁₋₁₀ heteroaliphatic having 1-5 heteroatoms, a 6-10 membered aryl ring, a 5-10 membered heteroaryl ring having 1-5 heteroatoms, and a 3-10 membered heterocyclic ring having 1-5 heteroatoms, or: two or more R′ groups are taken together with their intervening atoms to form an optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring having, in addition to the intervening atoms, 0-5 heteroatoms, wherein each monocyclic ring is independently an optionally substituted, saturated, partially saturated or aromatic 3-20 membered ring having 0-5 heteroatoms.
 9. The method of claim 8, wherein the aldol-dehydration product has the structure of formula P-2: R^(a)-L²-L¹-CH═CH—C(O)—C(O)OH,   P-2 or a salt thereof, wherein: R^(a) is R″ or —OR″, each of L¹ and L² is independently a covalent bond, or a bivalent, optionally substituted, linear or branched C₁₋₂₀ aliphatic or C₁₋₂₀ heteroaliphatic, wherein one or more methylene units are optionally and independently replaced by —C≡C—, —C(R″)₂—, -Cy-, —O—, —S—, —S—S—, —N(R″)—, —C(O)—, —C(S)—, —C(NR″)—, —C(O)N(R″)—, —N(R″)C(O)N(R″)—, —N(R″)C(O)O—, —S(O)—, —S(O)₂—, —S(O)₂N(R″)—, —C(O)S—, or —C(O)O—; -Cy- is a bivalent, optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring, wherein each monocyclic ring is independently an optionally substituted, saturated, partially saturated or aromatic 3-20 membered ring having 0-5 heteroatoms; each R″ is independently —R′, —C(O)R′, —CO₂R′, or —SO₂R′; R′ is hydrogen, or an optionally substituted group selected from C₁₋₁₀ aliphatic, C₁₋₁₀ heteroaliphatic having 1-5 heteroatoms, a 6-10 membered aryl ring, a 5-10 membered heteroaryl ring having 1-5 heteroatoms, and a 3-10 membered heterocyclic ring having 1-5 heteroatoms, or: two or more R′ groups are taken together with their intervening atoms to form an optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring having, in addition to the intervening atoms, 0-5 heteroatoms, wherein each monocyclic ring is independently an optionally substituted, saturated, partially saturated or aromatic 3-20 membered ring having 0-5 heteroatoms. 10-11. (canceled)
 12. The method of claim 9, wherein the alkene reduction product has the structure of formula P-3: R^(a)-L²-L¹-CH₂—CH₂—C(O)—C(O)OH,   P-3 or a salt thereof, wherein: R^(a) is R″ or —OR″, each of L¹ and L² is independently a covalent bond, or a bivalent, optionally substituted, linear or branched C₁₋₂₀ aliphatic or C₁₋₂₀ heteroaliphatic, wherein one or more methylene units are optionally and independently replaced by —C≡C—, —C(R″)₂—, -Cy-, —O—, —S—, —S—S—, —N(R″)—, —C(O)—, —C(S)—, —C(NR″)—, —C(O)N(R″)—, —N(R″)C(O)N(R″)—, —N(R″)C(O)O—, —S(O)—, —S(O)₂—, —S(O)₂N(R″)—, —C(O)S—, or —C(O)O—; -Cy- is a bivalent, optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring, wherein each monocyclic ring is independently an optionally substituted, saturated, partially saturated or aromatic 3-20 membered ring having 0-5 heteroatoms; each R″ is independently —R′, —C(O)R′, —CO₂R′, or —SO₂R′; R′ is hydrogen, or an optionally substituted group selected from C₁₋₁₀ aliphatic, C₁₋₁₀ heteroaliphatic having 1-5 heteroatoms, a 6-10 membered aryl ring, a 5-10 membered heteroaryl ring having 1-5 heteroatoms, and a 3-10 membered heterocyclic ring having 1-5 heteroatoms, or: two or more R′ groups are taken together with their intervening atoms to form an optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring having, in addition to the intervening atoms, 0-5 heteroatoms, wherein each monocyclic ring is independently an optionally substituted, saturated, partially saturated or aromatic 3-20 membered ring having 0-5 heteroatoms. 13-14. (canceled)
 15. The method of claim 12, wherein comprising converting a compound of formula P-3 or a salt thereof into a compound of formula P-4: R^(a)-L²-L¹-CH₂—CH₂—CH(OH)—C(O)OH,   P-4 or a salt thereof, wherein: R^(a) is R″ or —OR″, each of L¹ and L² is independently a covalent bond, or a bivalent, optionally substituted, linear or branched C₁₋₂₀ aliphatic or C₁₋₂₀ heteroaliphatic, wherein one or more methylene units are optionally and independently replaced by —C≡C—, —C(R″)₂—, -Cy-, —O—, —S—, —S—S—, —N(R″)—, —C(O)—, —C(S)—, —C(NR″)—, —C(O)N(R″)—, —N(R″)C(O)N(R″)—, —N(R″)C(O)O—, —S(O)—, —S(O)₂—, —S(O)₂N(R″)—, —C(O)S—, or —C(O)O—; -Cy- is a bivalent, optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring, wherein each monocyclic ring is independently an optionally substituted, saturated, partially saturated or aromatic 3-20 membered ring having 0-5 heteroatoms; each R″ is independently —R′, —C(O)R′, —CO₂R′, or —SO₂R′; R′ is hydrogen, or an optionally substituted group selected from C₁₋₁₀ aliphatic, C₁₋₁₀ heteroaliphatic having 1-5 heteroatoms, a 6-10 membered aryl ring, a 5-10 membered heteroaryl ring having 1-5 heteroatoms, and a 3-10 membered heterocyclic ring having 1-5 heteroatoms, or: two or more R′ groups are taken together with their intervening atoms to form an optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring having, in addition to the intervening atoms, 0-5 heteroatoms, wherein each monocyclic ring is independently an optionally substituted, saturated, partially saturated or aromatic 3-20 membered ring having 0-5 heteroatoms.
 16. (canceled)
 17. The method of claim 15, comprising converting a compound of formula P-4 or a salt thereof into a compound of formula P-5: R^(a)-L²-L¹-CH₂—CH₂—CH(OH)—C(O)—S—CoA,   P-5 or a salt thereof.
 18. (canceled)
 19. The method of claim 17, comprising converting a compound of formula P-5 or a salt thereof into a compound of formula P-6: R^(a)-L²-L¹-CH₂—CH═CH—C(O)—S—CoA,   P-6 or a salt thereof.
 20. (canceled)
 21. The method of claim 19, comprising converting a compound of formula P-6 or a salt thereof into a compound of formula P-7: R^(a)-L²-L¹-CH₂—CH₂—CH₂—C(O)—S—CoA,   P-7 or a salt thereof.
 22. (canceled)
 23. The method of claim 21, comprising converting a compound of formula P-7 or a salt thereof into a compound of formula P-8: R^(a)-L²-L¹-CH₂—CH₂—CH₂—C(O)—OH,   P-8 or a salt thereof.
 24. (canceled)
 25. The method of claim 23, comprising converting a compound of formula P-8, wherein L² is —CH₂-L^(2′)-, or a salt thereof into a compound of formula P-9: H—C(O)-L^(2′)-L¹-CH₂—CH₂—CH₂—C(O)—OH,   P-9 or a salt thereof, and/or converting a compound of formula P-9 or a salt thereof into a compound of formula P-10: HO—C(O)-L^(2′)-L¹-CH₂—CH₂—CH₂—C(O)—OH,   P-10 or a salt thereof, wherein: L^(2′) is a covalent bond, or a bivalent, optionally substituted, linear or branched C₁₋₁₉ aliphatic or C₁₋₁₉ heteroaliphatic, wherein one or more methylene units are optionally and independently replaced by —C≡C—, —C(R″)₂—, -Cy-, —O—, —S—, —S—S—, —N(R″)—, —C(O)—, —C(S)—, —C(NR″)—, —C(O)N(R″)—, —N(R″)C(O)N(R″)—, —N(R″)C(O)O—, —S(O)—, —S(O)₂—, —S(O)₂N(R″)—, —C(O)S—, or —C(O)O—. 26-28. (canceled)
 29. The method of claim 23, comprising converting a compound of formula P-8 or a salt thereof into a compound of formula P-9′: R^(a)-L²-L¹-CH₂—CH₂—CH₂—C(O)—H,   P-9′ or a salt thereof, and/or converting a compound of formula P-9′ or a salt thereof into a compound of formula P-10′: R^(a)-L²-L¹-CH₂—CH₂—CH₂—CH₂—OH,   P-10′ or a salt thereof. 30-32. (canceled)
 33. The method of claim 12, comprising converting a compound of formula P-3 or a salt thereof into a compound of formula P-4′: R^(a)-L²-L¹-CH₂—CH₂—C(O)—H,   P-4′ or a salt thereof, and/or converting a compound of formula P-4′ or a salt thereof into a compound of formula P-5′: R^(a)-L²-L¹-CH₂—CH₂—CH₂—OH,   P-5′ or a salt thereof. 34-65. (canceled)
 66. A preparation of a compound of formula P-1, P-2, P-3, P-4, P-4′, P-5, P-5′, P-6, P-7, P-8, P-9, P-9′, P-10, or P-10′, or a salt thereof, prepared by the method of claim 1, in which preparation is enriched for ¹⁴C isotope relative to that observed in a reference preparation of the compound, which reference preparation is prepared using fossil carbon source.
 67. A preparation of a polyester, a polyester polyol, a polyurethane, nylon 6, nylon 6,6, a polycarbonate diol, diacrylate ester, or diglycidyl ether, which preparation is manufactured using a preparation of claim
 66. 68. (canceled)
 69. An nucleic acid encoding the aldol-dehydration product biosynthesis polypeptides of claim 1, wherein the nucleic acid is optimized for expression in a microorganism.
 70. An engineered microbe that produces an aldol-dehydration product of an aliphatic aldehyde, the microbe comprising increased expression or activity of an aldol-dehydration product biosynthesis polypeptide, wherein: the carbonyl group of the aliphatic aldehyde is not conjugated to a alkenyl, alkynyl, or aromatic group; and the aldol-dehydration product is a compound comprising an aldehyde or ketone group and a double bond conjugated with the aldehyde or ketone group; or an engineered microbe that produces an alkene reduction product, the microbe comprising increased expression or activity of an alkene reduction product biosynthesis polypeptide, wherein: the alkene comprises a double bond conjugated to a carbonyl group; and a double bond conjugated to a carbonyl group in the alkene is reduced to a single bond to provide an alkene reduction product.
 71. (canceled)
 72. A culture, comprising a microbe of claim 70, and one or more compounds independently of formulae P-1, P-2, P-3, P-4, P-5, P-6, P-7, P-8, P-9, P-10, P-9′, P-10′, P-4′ or P-5′, or a salt thereof.
 73. (canceled) 